Signal transduction generally involves multivalent protein-protein interactions, which can produce various protein complexes and post-translational modifications. The reaction networks that characterize these interactions tend to be so large as to challenge conventional simulation procedures. To address this challenge, a kinetic Monte Carlo (KMC) method has been developed that can take advantage of a model specification in terms of reaction rules for molecular interactions. A set of rules implicitly defines the reactions that can occur as a result of the interactions represented by the rules. With the rule-based KMC method, explicit generation of the underlying chemical reaction network implied by rules is avoided. Here, we apply and extend this method to characterize the interactions of a trivalent ligand with a bivalent cell-surfacereceptor. This system is also studied experimentally. We consider the following kinetic models: an equivalent-site model, an extension of this model, which takes into account steric constraints on the configurations of receptoraggregates, and finally, a model that accounts for cyclic receptoraggregates. Simulation results for the equivalent-site model are consistent with an equilibrium continuum model. Using these models, we investigate the effects of steric constraints and the formation of cyclic aggregates on the kinetics and equilibria of small and large aggregate formation and the percolation phase transition that occurs in this system.

are sufficiently angular and rough in texture, thus ensuring mixture stability. A popular asphalt mixture design method called Superpave Level 1...would not pass either of the Superpave aggregate requirements. Table 18 Additional Characteristics for the Fine Fraction Abbreviated Common Name...CBR values when compacted wet of optimum. This is likely attributable to their relatively high permeabilities . For soaked CBR tests, the aggregates

Although the process of endocytosis of the low density lipoprotein (LDL) macromolecule and its receptor have been the subject of intense experimental research and modeling, there are still conflicting hypotheses and even conflicting data regarding the way receptors are transported to coated pits, the manner by which receptors are inserted before they aggregate in coated pits, and the display of receptors on the cell surface. At first it was considered that LDL receptors in human fibroblasts are inserted at random locations and then transported by diffusion toward coated pits. But experiments have not ruled out the possibility that the true rate of accumulation of LDL receptors in coated pits might be faster than predicted on the basis of pure diffusion and uniform reinsertion over the entire cell surface. It has been claimed that recycled LDL receptors are inserted preferentially in regions where coated pits form, with display occurring predominantly as groups of loosely associated units. Another mechanism that has been proposed by experimental cell biologists which might affect the accumulation of receptors in coated pits is a retrograde membrane flow. This is essentially linked to a polarized receptor insertion mode and also to the capping phenomenon, characterized by the formation of large patches of proteins that passively flow away from the regions of membrane exocytosis. In this contribution we calculate the mean travel time of LDL receptors to coated pits as determined by the ratio of flow strength to diffusion-coefficient, as well as by polarized-receptor insertion. We also project the resulting display of unbound receptors on the cell membrane. We found forms of polarized insertion that could potentially reduce the mean capture time of LDL receptors by coated pits which is controlled by diffusion and uniform insertion. Our results show that, in spite of its efficiency as a possible device for enhancement of the rate of receptor trapping, polarized

Although the process of endocytosis of the low density lipoprotein (LDL) macromolecule and its receptor have been the subject of intense experimental research and modeling, there are still conflicting hypotheses and even conflicting data regarding the way receptors are transported to coated pits, the manner by which receptors are inserted before they aggregate in coated pits, and the display of receptors on the cell surface. At first it was considered that LDL receptors in human fibroblasts are inserted at random locations and then transported by diffusion toward coated pits. But experiments have not ruled out the possibility that the true rate of accumulation of LDL receptors in coated pits might be faster than predicted on the basis of pure diffusion and uniform reinsertion over the entire cell surface. It has been claimed that recycled LDL receptors are inserted preferentially in regions where coated pits form, with display occurring predominantly as groups of loosely associated units. Another mechanism that has been proposed by experimental cell biologists which might affect the accumulation of receptors in coated pits is a retrograde membrane flow. This is essentially linked to a polarized receptor insertion mode and also to the capping phenomenon, characterized by the formation of large patches of proteins that passively flow away from the regions of membrane exocytosis. In this contribution we calculate the mean travel time of LDL receptors to coated pits as determined by the ratio of flow strength to diffusion-coefficient, as well as by polarized-receptor insertion. We also project the resulting display of unbound receptors on the cell membrane. We found forms of polarized insertion that could potentially reduce the mean capture time of LDL receptors by coated pits which is controlled by diffusion and uniform insertion. Our results show that, in spite of its efficiency as a possible device for enhancement of the rate of receptor trapping, polarized

In this work, the aggregation of charged liposomes induced by magnesium is investigated. Static and dynamic light scattering, Fourier-transform infrared spectroscopy, and cryotransmission electron microscopy are used as experimental techniques. In particular, multiple intracluster scattering is reduced to a negligible amount using a cross-correlation light scattering scheme. The analysis of the cluster structure, probed by means of static light scattering, reveals an evolution from surface fractals to mass fractals with increasing magnesium concentration. Cryotransmission electron microscopy micrographs of the aggregates are consistent with this interpretation. In addition, a comparative analysis of these results with those previously reported in the presence of calcium suggests that the different hydration energy between lipid vesicles when these divalent cations are present plays a fundamental role in the cluster morphology. This suggestion is also supported by infrared spectroscopy data. The kinetics of the aggregation processes is also analyzed through the time evolution of the mean diffusion coefficient of the aggregates.

The presence of gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) on gonadotrophs in the anterior pituitary (AP) is an important factor for reproduction control. However, little is known regarding GnRHR gene expression in gonadotrophs of cattle owing to the lack of an appropriate anti-GnRHR antibody. Therefore, an anti-GnRHR antibody for immunohistochemistry, flow cytometry, and immunocytochemistry assays was developed to characterize GnRHR gene expression in gonadotrophs. The anti-GnRHR antibody could suppress GnRH-induced LH secretion from cultured AP cells of cattle. The GnRHR, luteinizing hormone (LH), and follicle stimulating hormone (FSH) in the AP tissue was analyzed by fluorescence immunohistochemistry. The GnRHRs were aggregated on a limited area of the cell surface of gonadotrophs, possibly localized to lipid rafts. The LH secretion was stimulated with increasing amounts of GnRH; however, excessive concentrations (> 1 nM) resulted in a decrease in LH secretion. A novel method to purify gonadotrophs was developed using the anti-GnRHR antibody and fluorescence-activated cell sorting. Flow cytometric analysis using the anti-GnRHR antibody for cultured bovine AP cells, however, failed to support the hypothesis that GnRH induces GnRHR internalization and decreases GnRHR on the surface of GnRHR-positive AP cells. In contrast, immunocytochemistry using primary antibodies for cultured bovine AP cells showed that 10 nM (P < 0.05) and 100 nM (P < 0.01) GnRH, but not 0.01-1 nM GnRH, increased GnRHR in the cytoplasm of LH-positive cells. In conclusion, these data suggested that GnRHRs were aggregated on the surface of gonadotrophs and GnRHR inside gonadotrophs increased with elevated concentrations of GnRH.

Particle aggregation with simultaneous surface growth was modeled using a dynamic Monte Carlo method. The Monte Carlo algorithm begins in the particle inception zone and constructs aggregates via ensemble-averaged collisions between spheres and deposition of gaseous species on the sphere surfaces. Simulations were conducted using four scenarios. The first, referred to as scenario 0, is used as a benchmark and simulates aggregation in the absence of surface growth. Scenario 1 forces all balls to grow at a uniform rate while scenario 2 only permits them to grow once they have collided and stuck to each other. The last one is a test scenario constructed to confirm conclusions drawn from scenarios 0-2. The transition between the coalescent and the fully-developed fractal aggregation regimes is investigated using shape descriptors to quantify particle geometry. They are used to define the transition between the coalescent and fractal growth regimes. The simulations demonstrate that the morphology of aggregating particles is intimately related to both the surface deposition and particle nucleation rates.

Taking advantage of the extremely high dependence of surface tension on the concentration of amphiphilic molecules in aqueous solution, a new model based on the double equilibrium between free and aggregated molecules in the liquid phase and between free molecules in the liquid phase and those adsorbed at the air/liquid interface is presented and validated using literature data and fluorescence measurements. A key point of the model is the use of both the Langmuir isotherm and the Gibbs adsorption equation in terms of free molecules instead of the nominal concentration of the solute. The application of the model should be limited to non ionic compounds since it does not consider the presence of counterions. It requires several coupled nonlinear fittings for which we developed a software that is publicly available in our server as a web application. Using this tool, it is straightforward to get the average aggregation number of an amphiphile, the micellization free energy, the adsorption constant, the maximum surface excess (and so the minimum area per molecule), the distribution of solute in the liquid phase between free and aggregate species, and the surface coverage in only a couple of seconds, just by uploading a text file with surface tension vs concentration data and the corresponding uncertainties.

We have modeled platelet aggregation in a linear shear flow by accounting for two body collision hydrodynamics, platelet activation and receptor biology. Considering platelets and their aggregates as unequal-sized spheres with DLVO interactions (psi(platelet) = -15 mV, Hamaker constant = 10(-19) J), detailed hydrodynamics provided the flow field around the colliding platelets. Trajectory calculations were performed to obtain the far upstream cross-sectional area and the particle flux through this area provided the collision frequency. Only a fraction of platelets brought together by a shearing fluid flow were held together if successfully bound by fibrinogen cross-bridging GPIIb/IIIa receptors on the platelet surfaces. This fraction was calculated by modeling receptor-mediated aggregation using the formalism of Bell (Bell, G. I. 1979. A theoretical model for adhesion between cells mediated by multivalent ligands. Cell Biophys. 1:133-147) where the forward rate of bond formation dictated aggregation during collision and was estimated from the diffusional limited rate of lateral association of receptors multiplied by an effectiveness factor, eta, to give an apparent rate. For a value of eta = 0.0178, we calculated the overall efficiency (including both receptor binding and hydrodynamics effects) for equal-sized platelets with 50,000 receptors/platelet to be 0.206 for G = 41.9 s(-1), 0.05 for G = 335 s(-1), and 0.0086 for G = 1920 s(-1), values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. From our analysis, we predict that bond formation proceeds at a rate of approximately 0.1925 bonds/microm2 per ms, which is approximately 50-fold slower than the diffusion limited rate of association. This value of eta is also consistent with a colloidal stability of unactivated platelets at low shear rates. Fibrinogen was calculated to mediate aggregation quite efficiently at low shear rates but not at

Glycopeptides were removed by trypsinization from the surface of baby hamster kidney cells (line BHK21-C13), digested by pronase and separated into 2 fractions by exclusion chromatography. The addition of small amounts of either glycopeptide fraction to shaken suspensions of lightly trypsinzied cells inhibited their rapid aggregation, but one fraction was more active than the other and in higher concentrations it was able to inhibit aggregation completely. After this fraction was purified by high-voltage electrophoresis one subfraction also inhibited aggregation. The effect of the glycopeptides increased following their pretreatment with neuraminidase, but preincubation with periodiate or galactose oxidase destroyed all activity. Galactose oxidase also inhibited cell aggregation directly. Similar glycopeptides from virus-transformed BHK21 cells, oligosaccharides and intact and desialysed human urinary glycoproteins had comparatively little or no effect on BHK21 cell aggregation. The results suggest terminal beta-galactosides and possible alpha-galactosides, and to some extent a particular substructure of cell surface heteroglycans are necessary for their inhibitory activity. The parent, plasma membrane of glycoproteins might serve as adhesive binding sites in cell cohesion, but some evidence indicates cell surface sialyl- and galactosyltransferases may not ordinarily act as their complementary binding receptors.

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another, (2) can strongly influence the ability of tissues to interact with biomaterials, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion, but also by manipulation of key ECM molecules such as FN and 4) correlates with invasive potential of lung cancer, fibrosarcoma, brain tumor and prostate tumor cell lines. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

Aggregation and autophosphorylation of the insulin receptor-protein kinase, from cultured 3T3-L1 adipocytes, were studied in the presence of cationic polyamino acids. Poly-L-lysine and poly-L-arginine produced the following effects with the purified receptor: first, the autophosphorylation rate was increased by polycations. Half-maximal stimulation was proportional to polymer length. The rate enhancement was greater at lower ATP concentrations. Second, near-endpoint (equilibrium) autophosphorylation was greater in the presence of the polycations. Polycations inhibited the reverse reaction: ADP + phosphoreceptor yielding ATP + aporeceptor. Third, the (32P)phosphopeptides generated by trypsin digestion of the 32P-beta-subunit, showed that no new autophosphorylation sites resulted from the presence of polycations. Fourth, the polycations, but not insulin, promoted receptoraggregation, and phosphoreceptor aggregated more readily than aporeceptor. Insulin receptor enriched through the wheat germ agglutinin eluate step was compared with purified receptor. Higher concentrations of poly-L-arginine were required to stimulate autophosphorylation and to promote aggregation. Finally, several polycation-dependent substrates present in the wheat germ agglutinin eluate co-aggregated with the insulin receptor. Polycation-stimulated receptor autophosphorylation is linked to a lower KM,app for ATP, but substrate phosphorylation may require the aggregation.

Abstract The complex, multistep aggregation kinetic and structural behavior of human recombinant interleukin-1 receptor antagonist (IL-1ra) was revealed and characterized by spectral probes and techniques. At a certain range of protein concentration (12–27 mg/mL) and temperature (44–48°C), two sequential aggregation kinetic transitions emerge, where the second transition is preceded by a lag phase and is associated with the main portion of the aggregated protein. Each kinetic transition is linked to a different type of aggregate population, referred to as type I and type II. The aggregate populations, isolated at a series of time points and analyzed by Fourier-transform infrared spectroscopy, show consecutive protein structural changes, from intramolecular (type I) to intermolecular (type II) β-sheet formation. The early type I protein spectral change resembles that seen for IL-1ra in the crystalline state. Moreover, Fourier-transform infrared data demonstrate that type I protein assembly alone can undergo a structural rearrangement and, consequently, convert to the type II aggregate. The aggregated protein structural changes are accompanied by the aggregate morphological changes, leading to a well-defined population of interacting spheres, as detected by scanning electron microscopy. A nucleation-driven IL-1ra aggregation pathway is proposed, and assumes two major activation energy barriers, where the second barrier is associated with the type I → type II aggregate structural rearrangement that, in turn, serves as a pseudonucleus triggering the second kinetic event. PMID:19134476

To examine the role of the hyaluronate receptor in cell to cell adhesion, the authors have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks its ability to interact with hyaluronate. In the first set of experiments, they investigated the spontaneous aggregation of SV-3T3 cells, which involves two distinct mechanisms, one of which is dependent upon the presence of divalent cation and the other is independent. The divalent cation-independent aggregation was found to be completely inhibited by both intact and Fab fragments of the K-3 MAb. In contrast, the K-3 MAb had no effect on the divalent cation-dependent aggregation of cells. In a second set of experiments, we examined alveolar macrophages. The presence of hyaluronate receptors on alveolar macrophages was demonstrated by the fact that detergent extracts of these cells could bind ({sup 3})hyaluronate, and this binding was blocked by the K-3 MAb. Immunoblot analysis of alveolar macrophages showed that the hyaluronate receptor had a M{sub r} of 99,500, which is considerably larger than the 85,000 M{sub r} for that on BHK cells. When hyaluronate was added to suspensions of alveolar macrophages, the cells were induced to aggregate. This effect was inhibited by the K-3 MAb, suggesting that the hyaluronate-induced aggregation was mediated by the receptor.

We carried out parallel experiments first on the slow clinostat and then in space-flight to examine the effects of altered gravity on the aggregation of the nicotinic acetylcholine receptors and the structure of the cytoskeleton in cultured Xenopus embryonic muscle cells. By examining the concordance between results from space flight and the clinostat, we tested whether the slow clinostat is a relevant simulation paradigm. Space-flown cells showed marked changes in the distribution and organization of actin filaments and had a reduced incidence of acetylcholine receptoraggregates at the site of contact with polystyrene beads. Similar effects were found after clinostat rotation. The sensitivity of synaptic receptoraggregation and cytoskeletal morphology suggests that in the microgravity of space cell behavior may be importantly altered.

Dopamine receptors are a class of metabotropic G protein-coupled receptors. Plasma membrane expression is a key determinant of receptor signaling, and one that is regulated both by extra and intracellular cues. Abnormal dopamine receptor signaling is implicated in several neuropsychiatric disorders, including schizophrenia and attention deficit hyperactivity disorder, as well as drug abuse. Here, we describe in detail the application of two complementary applications of protein biotinylation and enzyme-linked immunoabsorbent assay (ELISA) for detecting and quantifying levels of dopamine receptors expressed on the cell surface. In the biotinylation method, cell surfacereceptors are labeled with Sulfo-NHS-biotin. The charge on the sulfonyl facilitates water solubility of the reactive biotin compound and prevents its diffusion across the plasma membrane. In the ELISA method, surface labeling is achieved with antibodies specific to extracellular epitopes on the receptors, and by fixing the cells without detergent such that the plasma membrane remains intact.

Dopamine receptors are a class of metabotropic G protein-coupled receptors. Plasma membrane expression is a key determinant of receptor signaling, and one that is regulated both by extra and intracellular cues. Abnormal dopamine receptor signaling is implicated in several neuropsychiatric disorders, including schizophrenia and attention deficit hyperactivity disorder, as well as drug abuse. Here, we describe in detail the application of two complementary applications of protein biotinylation and enzyme-linked immunoabsorbant assay (ELISA) for detecting and quantifying levels of dopamine receptors expressed on the cell surface. In the biotinylation method, cell surfacereceptors are labeled with Sulfo-NHS-biotin. The charge on the sulfonyl facilitates water solubility of the reactive biotin compound and prevents its diffusion across the plasma membrane. In the ELISA method, cells surface labeling is achieved with antibodies specific to extracellular epitopes on the receptors, and by fixing the cells without detergent such that the plasma membrane remains intact. PMID:23296774

Dictyostelium discoideum, a eukaryotic amoeba of the cellular slime mold family, provides an interesting paradigm in developmental biology. During development, hundreds of thousands of cells aggregate to form a multicellular aggregate. Aggregation is mediated by chemotaxis and chemical signaling. Waves of adenosine 3'-5' cyclic monophosphate (cAMP) propagate through the monolayer and provide transient gradients for chemotaxis. The author has used a reversible inhibitor of the cAMP signaling response to demonstrate that adaptation to cAMP is independent of the activation of the adenylate cyclase and therefore is not caused by the rise in intracellular cAMP. Next, it is shown that adenosine inhibits the cAMP signaling response. Inhibition is rapid, reversible, and depends on the cAMP stimulus concentration. Then the specificity of the cAMP receptors which mediates signaling is determined and compared with the receptors which mediate chemotaxis, the cGMP response, and cAMP binding antagonism. The cAMP surfacereceptor has been identified by photoaffinity labeling intact cells with (/sup 32/P)-8-N/sub 3/-cAMP using an ammonium sulfate binding stabilization technique. The photoactivated ligand specifically labels a polypeptide, localized to the membrane fraction, which migrates as a closely spaced doublet on SDS Page.

Fluorescence resonance energy transfer between epidermal growth factor (EGF) molecules, labeled with fluorescent reporter groups, was used as a monitor for EGF receptor-receptor interactions in plasma membranes isolated from human epidermoid A431 cells. Epidermal growth factor molecules labeled at the amino terminus with fluorescein isothiocyanate served as donor molecules in these energy transfer measurements, while EGF molecules labeled with eosin isothiocyanate at the amino terminus served as the energy acceptors. Both of these derivatives were shown to be active in binding to membrane receptors and in the activation of the endogenous receptor/tyrosine kinase activity. We found that membranes in the absence of added metal ion activators showed relatively little energy transfer (approximately 10% donor quenching) between the labeled growth factors. However, divalent metal ion activators of the EGF receptor/tyrosine kinase caused a significant increase in the extent of energy transfer between the labeled EGF molecules. Specifically, in the presence of 20 mM MgCl2, the extent of quenching of the donor fluorescence increased to 25% (from 10% in the absence of metal), while in the presence of 4 mM MnCl2, the extent of energy transfer was increased still further to 40-50%. The addition of an excess of EDTA resulted in the reversal of the observed energy transfer to basal levels. The increased energy transfer in the presence of these divalent cations correlated well with the ability of these metals to stimulate the EGF receptor/tyrosine kinase activity. However, the extent of receptor-receptor interactions measured by energy transfer was independent of receptor autophosphorylation. Overall, these results suggest that conditions under which the EGF receptor is primed to be active as a tyrosine kinase, within a lipid milieu, result in an increased aggregation of the receptor.

This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT1)-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT1-receptor antagonist-stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT1-receptor antagonists on platelet adhesion to collagen and thromboxane A2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 micro mol/L, which was attenuated by NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. The angiotensin II AT1-receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT1-receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N(G)-nitro-L-arginine methyl ester. Neither the AT2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A2/prostaglandin H2 receptor antagonist SQ29,548 had any effect on angiotensin II AT1-receptor antagonist-stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT1-receptor antagonists.

The CCN family (CYR61; CTGF; NOV; CCN1-6; WISP1-3) of matricellular proteins in mammals is comprised of six homologous members that play important roles in development, inflammation, tissue repair, and a broad range of pathological processes including fibrosis and cancer. Despite considerable effort to search for a high affinity CCN-specific receptor akin to growth factor receptors, no such receptor has been found. Rather, CCNs bind several groups of multi-ligand receptors as characteristic of other matricellular proteins. The most extensively documented among CCN-binding receptors are integrins, including αvβ3, αvβ5, α5β1, α6β1, αIIbβ3, αMβ2, and αDβ2, which mediate diverse CCN functions in various cell types. CCNs also bind cell surface heparan sulfate proteoglycans (HSPGs), low density liproprotein receptor-related proteins (LRPs), and the cation-independent mannose-6-phosphate (M6P) receptor, which are endocytic receptors that may also serve as co-receptors in cooperation with other cell surfacereceptors. CCNs have also been reported to bind FGFR-2, Notch, RANK, and TrkA, potentially altering the affinities of these receptors for their ligands. The ability of CCNs to bind a multitude of receptors in various cell types may account for the remarkable versatility of their functions, and underscore the diverse signaling pathways that mediate their activities.

The research effort in the Waychunas group is focused on the characterization and measurement of processes at the mineral-water interfaces specifically related to the onset of precipitation. This effort maps into one of the main project groups with the Penn State University EMSI (CEKA) known as PIG (Precipitation Interest Group), and involves collaborations with several members of that group. Both synchrotron experimentation and technique development are objectives, with the goals of allowing precipitation from single molecule attachment to sub-monolayer coverage to be detected and analyzed. The problem being addressed is the change in reactivity of mineral interfaces due to passivation or activation by precipitates or sorbates. In the case of passivation, fewer active sites may be involved in reactions with environmental fluids, while in the activated case the precipitate may be much more reactive than the substrate, or result in the creation of a higher density of active sites. We approach this problem by making direct measurements of several types of precipitation reactions: iron-aluminum oxide formation on quartz and other substrates from both homogeneous (in solution) nucleation, and heterogeneous (on the surface) nucleation; precipitation and sorption of silicate monomers and polymers on Fe oxide surfaces; and development of grazing-incidence small angle x-ray scattering (GISAXS) as a tool for in-situ measurement of precipitate growth, morphology and aggregation. We expect that these projects will produce new fundamental information on reactive interface growth, passivation and activation, and be applicable to a wide range of environmental interfaces.

We studied two particular biomedical systems which exhibit collective molecular behavior. One is clustering of tumor necrosis factor receptor I (TNFR1), and another is β-sheet folding and aggregation. Receptor clustering has been shown to be a crucial step in many signaling events but its biological meaning has not been adequately addressed. Here, via a simple lattice model, we show how cells use this clustering machinery to enhance sensitivity as well as robustness. On the other hand, intracellular deposition of aggregated protein rich in β-sheet is a prominent cytopathological feature of most neurodegenerative diseases. How this aggregation occurs and how it responds to therapy is not completely understood. Here, we started from a reconstruction of the H-bond potential and carry out a full investigation of β-sheet thermodynamics as well as kinetics. We show that β-sheet aggregation is most likely due to molecular stacking and found that the minimal length of an aggregate mutant polymer corresponds well with the number observed in adult Huntington's disease. We have also shown that molecular agents such as dendrimers might fail at high-dose therapy; instead, a potential therapy strategy is to block β-turn formation. Our predictions can be used for future experimental tests and clinical trials.

Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.

Using epifluorescence microscopy and image analysis, we have quantitatively described the frequency, size, and spatial distribution of bacterial aggregates on leaf surfaces of greenhouse-grown bean plants inoculated with the plant-pathogenic bacterium Pseudomonas syringae pv. syringae strain B728a. Bacterial cells were not randomly distributed on the leaf surface but occurred in a wide range of cluster sizes, ranging from single cells to over 10(4) cells per aggregate. The average cluster size increased through time, and aggregates were more numerous and larger when plants were maintained under conditions of high relative humidity levels than under dry conditions. The large majority of aggregates observed were small (less than 100 cells), and aggregate sizes exhibited a strong right-hand-skewed frequency distribution. While large aggregates are not frequent on a given leaf, they often accounted for the majority of cells present. We observed that up to 50% of cells present on a leaf were located in aggregates containing 10(3) cells or more. Aggregates were associated with several different anatomical features of the leaf surface but not with stomates. Aggregates were preferentially associated with glandular trichomes and veins. The biological and ecological significance of aggregate formation by epiphytic bacteria is discussed.

Densely packed surface fractal aggregates form in systems with high local volume fractions of particles with very short diffusion lengths, which effectively means that particles have little space to move. However, there are no prior mathematical models, which would describe scattering from such surface fractal aggregates and which would allow the subdivision between inter- and intraparticle interferences of such aggregates. Here, we show that by including a form factor function of the primary particles building the aggregate, a finite size of the surface fractal interfacial sub-surfaces can be derived from a structure factor term. This formalism allows us to define both a finite specific surface area for fractal aggregates and the fraction of particle interfacial sub-surfaces at the perimeter of an aggregate. The derived surface fractal model is validated by comparing it with an ab initio approach that involves the generation of a "brick-in-a-wall" von Koch type contour fractals. Moreover, we show that this approach explains observed scattering intensities from in situ experiments that followed gypsum (CaSO4 ṡ 2H2O) precipitation from highly supersaturated solutions. Our model of densely packed "brick-in-a-wall" surface fractal aggregates may well be the key precursor step in the formation of several types of mosaic- and meso-crystals.

Synaptic pathology in Alzheimer's disease brains is thought to involve soluble Aβ42 peptide. Here, sterile incubation in PBS caused small Aβ42 oligomer formation as well as heterogeneous, 6E10-immunopositive aggregates of 80-100 kDa. High molecular weight aggregates (H-agg) formed in a time-dependent manner over an extended 30-day period. Interestingly, an inverse relationship between dimeric and H-agg formation was more evident when incubations were performed at 37°C as compared to 23°C, thus providing an experimental strategy with which to address synaptic compromise produced by the different Aβ aggregates. H-agg species formed faster and to higher levels at 37°C compared to 23°C, and the two aggregate preparations were evaluated in hippocampal slice cultures, a sensitive system for monitoring synaptic integrity. Applied daily at 80-600 nM for 7 days, the Aβ42 preparations caused dose-dependent and aggregation-dependent declines in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits as well as in presynaptic components. Unlike the synaptic effects, Aβ42 induced only trace cellular degeneration that was CA1 specific. The 37°C preparation was less effective at decreasing synaptic markers, corresponding with its reduced levels of Aβ42 monomers and dimers. Aβ42 dimers decayed significantly faster at 37°C than 23°C, and more rapidly than monomers at either temperature. These findings indicate that Aβ42 can self-aggregate into potent synaptotoxic oligomers as well as into larger aggregates that may serve to neutralize the toxic formations. These results will add to the growing debate concerning whether high molecular weight Aβ complexes that form amyloid plaques are protective through the sequestration of oligomeric species. PMID:21978994

The aggregation of proteins became one of the major challenges in the development of biopharmaceu-ticals since the formation of aggregates can affect drug quality and immunogenicity. However, aggregation mechanisms are highly complex and the investigation requires cost, time, and material intensive experi-mental effort. In the present work, the predictive power of protein characteristics for the phase behavior of three different proteins which are very similar in size and structure was studied. In particular, the surface hydrophobicity, zeta potential, and conformational flexibility of human lysozyme, lysozyme from chicken egg white, and α-lactalbumin at pH 3, 5, 7, and 9 were assessed and examined for correlation with experimental stability studies focusing on protein phase behavior induced by sodium chloride and ammonium sulfate. The molecular dynamics (MD) simulation based study of the conformational flexibility without precipitants was able to identify highly flexible protein regions which could be associated to the less regular secondary structure elements and random coiled and terminal regions in particular. Conformational flex-ibility of the entire protein structure and protein surface hydrophobicity could be correlated to differing aggregation propensities among the studied proteins and could be identified to be applicable for predic-tion of protein phase behavior in aqueous solution without precipitants. For prediction of protein phase behavior and aggregation propensity in aqueous solution with precipitants, protein flexibility was further studied in dependency of salt concentration and species by means of human lysozyme. Even though the results of the salt dependent MD simulations could not be shown to be sufficient for prediction of salt depending phase behavior, this study revealed a more pronounced destabilizing effect of ammonium sulfate in comparison to sodium chloride and thus, was found to be in good agreement with theoretical considerations

Platelet P2Y12 is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma, we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises 10 µM ADP-induced platelet aggregation in a concentration-dependent manner with an IC50 value of ~250 µM. An eight-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2S-UTP), with an IC50 of 30 µM. The 4-thio analogue (4S-UTP) with an IC50 of 7.5 µM was 33-fold more effective. A three-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptor was verified by P2Y12 receptor binding and cyclic AMP (cAMP) assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that may be useful for therapeutic intervention. PMID:28146050

We present a study of the dynamics of protein aggregation using a common path heterodyne Bloch surface wave sensing scheme. We demonstrate the ability to detect, during thermal incubation, the early events linked to the aggregation of proteins related to conformational diseases. Alzheimer's amyloid-β 1-42 is used to demonstrate the efficiency of the method. A model based on elementary interactions is shown to describe accurately the aggregation process. The described sensing scheme is sensitive to the early events of the aggregation process. is hence proposed as a method for the detection of early stages of the evolution of conformational diseases.

Some Lactobacillus brevis strains were found to aggregate upon the addition of xylan after screening for lactic acid bacteria that interact with plant materials. The S-layer proteins of cell surface varied among the strains. The strains that displayed xylan-mediated aggregation retained its ability even after the removal of S-layer proteins. L. brevis had negative zeta potentials. A correlation between the strength of aggregation and zeta potential was not observed. However, partial removal of S-layer proteins resulted in decreases in the electric potential and aggregation ability of some strains. Therefore, xylan-mediated aggregation of L. brevis was considered to be caused by an electrostatic effect between the cells and xylan. L. brevis also aggregated in the presence of mucin, and the strengths of aggregation among the strains were similar to that induced by xylan. Thus, xylan- and mucin-mediated L. brevis aggregation was supposed to be caused by a similar mechanism.

There are a vast number of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), associated with the rearrangement of specific proteins to non-native conformations that promotes aggregation and deposition within tissues and/or cellular compartments. These diseases are commonly classified as protein-misfolding or amyloid diseases. The interaction of these proteins with liquid/surface interfaces is a fundamental phenomenon with potential implications for protein-misfolding diseases. Kinetic and thermodynamic studies indicate that significant conformational changes can be induced in proteins encountering surfaces, which can play a critical role in nucleating aggregate formation or stabilizing specific aggregation states. Surfaces of particular interest in neurodegenerative diseases are cellular and subcellular membranes that are predominately comprised of lipid components. The two-dimensional liquid environments provided by lipid bilayers can profoundly alter protein structure and dynamics by both specific and non-specific interactions. Importantly for misfolding diseases, these bilayer properties can not only modulate protein conformation, but also exert influence on aggregation state. A detailed understanding of the influence of (sub)cellular surfaces in driving protein aggregation and/or stabilizing specific aggregate forms could provide new insights into toxic mechanisms associated with these diseases. Here, we review the influence of surfaces in driving and stabilizing protein aggregation with a specific emphasis on lipid membranes. PMID:23459674

surfaced pavements . The performance of aggregate-surfaced pavements is greatly affected by climate conditions and the presence of water, especially in... pavements . The study was sponsored by the US Air Force. The work was performed by the Airfields and Pavements Branch (GM-A) of the Engineering...scenarios. The current flexible pavement design criteria require corrections that account for the different performance and degradation of aggregate

We present here the effect of firefly luciferase surface charge saturation and the presence of some additives on its thermal-induced aggregation. Three mutants of firefly luciferase prepared by introduction of surface Arg residues named as 2R, 3R and 5R have two, three and five additional arginine residues substituted at their surface compared to native luciferase; respectively. Turbidimetric study of heat-induced aggregation indicates that all three mutants were reproducibly aggregated at higher rates relative to wild type in spite of their higher thermostability. Among them, 2R had most evaluated propensity to heat-induced aggregation. Therefore, the hydrophilization followed by appearing of more substituted arginine residues with positive charge on the firefly luciferase surface was not reduced its thermal aggregation. Nevertheless, at the same condition in the presence of charged amino acids, e.g. Arg, Lys and Glu, as well as a hydrophobic amino acid, e.g. Val, the heat-induced aggregation of wild type and mutants of firefly luciferases was markedly decelerated than those in the absence of additives. On the basis of obtained results it seems, relinquishment of variety in charge of amino acid side chains, they via local interactions with proteins cause to decrease rate and extent of their thermal aggregation.

The dissolution of CaCO(3) is one of the ways ocean acidification can, potentially, greatly affect the ballast of aggregates. A diminution of the ballast could reduce the settling speed of aggregates, resulting in a change in the carbon flux to the deep sea. This would mean lower amounts of more refractory organic matter reaching the ocean floor. This work aimed to determine the effect of ocean acidification on the ballast of sinking surfaceaggregates. Our hypothesis was that the decrease of pH will increase the dissolution of particulate inorganic carbon ballasting the aggregates, consequently reducing their settling velocity and increasing their residence time in the upper twilight zone. Using a new methodology for simulation of aggregate settling, our results suggest that future pCO(2) conditions can significantly change the ballast composition of sinking aggregates. The change in aggregate composition had an effect on the size distribution of the aggregates, with a shift to smaller aggregates. A change also occurred in the settling velocity of the particles, which would lead to a higher residence time in the water column, where they could be continuously degraded. In the environment, such an effect would result in a reduction of the carbon flux to the deep-sea. This reduction would impact those benthic communities, which rely on the vertical flow of carbon as primary source of energy.

We study the aggregation kinetics of two elastomer colloids with similar bulk polymer properties but with different surface charge groups in order to understand the role of the surface properties in particle coalescence during aggregation. It is confirmed that clusters of the elastomer particles stabilized purely by ionic surfactants coalesce in both reaction-limited and diffusion-limited aggregation (RLCA and DLCA) regimes and that the coalescence is independent of the coagulant type. On the other hand, clusters formed by elastomer particles stabilized by charged polymer end groups, which are fixed on the particle surface, are fractal objects with a fractal dimension of 1.7 in the DLCA and 2.1 in the RLCA regime. This indicates insignificant cluster coalescence during aggregation, most likely due to a hindrance effect of the fixed charges.

The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the

Adsorption of a short-chain nonionic amphiphile (C6E3) at the surface of mesoporous silica glass (CPG-10) was studied by a combination of adsorption measurements and mesoscale simulations. Adsorption measurements covering a wide composition range of the C6E3 + water system show that no adsorption occurs up to the critical micelle concentration (cmc), at which a sharp increase of adsorption is observed that is attributed to ad-micelle formation at the pore walls. Intriguingly, as the concentration is increased further, the surface excess of the amphiphile begins to decrease and eventually becomes negative, which corresponds to preferential adsorption of water rather than amphiphilemore » at high amphiphile concentrations. The existence of such a surface-azeotropic point has not previously been reported in the surfactant adsorption field. Dissipative particle dynamics (DPD) simulations were performed to reveal the structural origin of this transition from aggregative adsorption to surface depletion. Finally, the simulations indicate that this transition can be attributed to the repulsive interaction between head groups, causing amphiphilic depletion in the region around the corona of the surface micelles.« less

We investigated the whole cell distribution of the platelet membrane receptor for fibrinogen in surface-activated human platelets. Fibrinogen-labeled colloidal gold was used in conjunction with platelet whole mount preparations to visualize directly the fibrinogen receptor. Unstimulated platelets fail to bind fibrinogen, and binding was minimal in the stages of activation immediately following adhesion. The amount of fibrinogen bound per platelet increased rapidly during the shape changes associated with surface activation until 7,600 +/- 500 labels were present at saturation. Maximal binding of fibrinogen was followed by receptor redistribution. During the early stages of spreading, fibrinogen labels were uniformly distributed over the entire platelet surface, including pseudopodia, but the labels become progressively centralized as the spreading process continued. In well spread platelets, labels were found over the central regions, whereas peripheral areas were cleared of receptors. Receptor redistribution during spreading was accompanied by cytoskeletal reorganization such that a direct correlation was seen between the development of specific ultrastructural zones and the distribution of surfacereceptor sites suggesting a link between the surfacereceptors and the cytoskeleton. The association of fibrinogen receptors with contractile elements of the cytoskeleton, which permits coordinated receptor centralization, is important to the understanding of the role of fibrinogen in normal platelet aggregation and clot retraction. PMID:6088559

The effect of medium pH on conidial aggregation during submerged cultivation of Aspergillus niger is considered to originate from the electrostatic surface properties of the spores. As previously shown, these properties are greatly influenced by the presence of a melanin-containing surface coating covering the outer spore wall layer. The present study was designed to elucidate the impact of such a coating on the spores' surface potential and their electrostatic repulsion under acidic conditions. A Poisson-Boltzmann model was proposed and potential profiles across the surface coating of noninteracting and interacting spores were calculated. The surface potentials thus obtained were in line with the observed pH dependence of the zeta potential. This dependence was consistent with the outcome of aggregation experiments. Apparently contradictory results regarding the zeta potential and the aggregation behavior of the spores were obtained when the ionic strength was varied. However, both of these observations could be explained by the model.

Effect of side chain length on the molecular aggregation states and surface properties of poly(fluoroalkyl acrylate)s [PFA-Cy, where y is fluoromethylene number in Rf group] thin films were systematically investigated. Spin-coated PFA-Cy thin films were characterized by static and dynamic contact angle measurements, X-ray photoelectron spectroscopy (XPS), and grazing- incidence X-ray diffraction (GIXD). The receding contact angles showed small values for PFA-Cy with short side chain (y<=6) and increased above y>=8. GIXD revealed that fluoroalkyl side chain of PFA-Cy with y>=8 was crystallized and formed ordered structures at the surface region as well as bulk one. These results suggest that water repellent mechanism of PFA-Cy can be attributed to the presence of highly ordered fluoroalkyl side chains at the outermost surfaces. The results of XPS in the dry and hydrated states and contact angle measurement in water support the mechanism of lowering contact angle for water by exposure of carbonyl group to the water interface through reorientation of short fluoroalkyl chains. The surface nanotextured PFA-C8 through imprinting of anodic aluminum oxide mold showed extremely high hydrophobicity as well as high oleophobicity.

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial

Aggregation measurement of washed human erythrocytes (RBC) were carried out in a NaCl-PBS solution under laminar shear conditions. Artificial aggregation of fresh and stored erythrocytes was caused by decreased pH and reduced ionic strength and characterized by collision efficiency alpha. Generally, the collision efficiency alpha of stored erythrocytes rises with the increased storage time. Such an aggregation technique might be useful to detect and quantify changes of the membrane and/or the surface structure due to aging and/or storage.

Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of approximately 2.3 X 10(-9) cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptoraggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusion-mediated aggregation. PMID:3170634

Homocysteine (Hcy) is an excitotoxic amino acid. It is potentially possible to prevent Hcy-induced toxicity, including haemostatic impairments, by antagonizing glutaminergic receptors. Using impedance aggregometry with arachidonate and collagen as platelet agonists, we tested whether the blockade of platelet NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and kainate receptors with their inhibitors: MK-801 (dizocilpine hydrogen maleate, [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine), CNQX (7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile) and UBP-302 (2-{[3-[(2S)-2-amino-2-carboxyethyl]-2,6-dioxo-3,6-dihydropyrimidin 1(2H)-yl]methyl}benzoic acid) may hamper Hcy-dependent platelet aggregation. All the tested compounds significantly inhibited Hcy-augmented aggregation of blood platelets stimulated either with arachidonate or collagen. Hcy stimulated the generation of superoxide anion in whole blood samples in a concentration-dependent manner; however, this process appeared as independent on ionotropic glutamate receptors, as well as on NADPH oxidase and protein kinase C, and was not apparently associated with the extent of either arachidonate- or collagen-dependent platelet aggregation. Moreover, Hcy acted as a significant fluidizer of surface (more hydrophilic) and inner (more hydrophobic) regions of platelet membrane lipid bilayer, when used at the concentration range from 10 to 50 µmol/l. However, this effect was independent on the Hcy action through glutamate ionotropic receptors, since there was no effects of MK-801, CNQX or UBP-302 on Hcy-mediated membrane fluidization. In conclusion, Hcy-induced changes in whole blood platelet aggregation are mediated through the ionotopic excitotoxic receptors, although the detailed mechanisms underlying such interactions remain to be elucidated.

The purpose of this study was to explore the use of surface-enhanced Raman spectroscopy (SERS) to image the distribution of epidermal growth factor receptor (EGFR) in cells. To accomplish this task, 30-nm gold nanoparticles (AuNPs) tagged with antibodies to EGFR (1012 per mL) were incubated with cells (106 per mL) of the A431 human epidermoid carcinoma and normal human bronchial epithelial cell lines. Using the 632.8-nm excitation line of a He-Ne laser, Raman spectroscopy measurements were performed using a point mapping scheme. Normal cells show little to no enhancement. SERS signals were observed inside the cytoplasm of A431 cells with an overall enhancement of 4 to 7 orders of magnitude. Raman intensity maps of the 1450 and 1583 cm-1 peaks correlate well with the expected distribution of EGFR and AuNPs, aggregated following uptake by endosomes and lysosomes. Spectral features from tyrosine and tryptophan residues dominate the SERS signals.

Protein aggregation plays a critical role in the pathogenesis of neurodegenerative diseases, and the mechanism of its progression is poorly understood. Here, we examine the structural and dynamic characteristics of transiently evolving protein aggregates under ambient conditions by directly probing protein surface water diffusivity, local protein segment dynamics, and interprotein packing as a function of aggregation time, along the third repeat domain and C terminus of Δtau187 spanning residues 255–441 of the longest isoform of human tau. These measurements were achieved with a set of highly sensitive magnetic resonance tools that rely on site-specific electron spin labeling of Δtau187. Within minutes of initiated aggregation, the majority of Δtau187 that is initially homogeneously hydrated undergoes structural transformations to form partially structured aggregation intermediates. This is reflected in the dispersion of surface water dynamics that is distinct around the third repeat domain, found to be embedded in an intertau interface, from that of the solvent-exposed C terminus. Over the course of hours and in a rate-limiting process, a majority of these aggregation intermediates proceed to convert into stable β-sheet structured species and maintain their stacking order without exchanging their subunits. The population of β-sheet structured species is >5% within 5 min of aggregation and gradually grows to 50–70% within the early stages of fibril formation, while they mostly anneal block-wisely to form elongated fibrils. Our findings suggest that the formation of dynamic aggregation intermediates constitutes a major event occurring in the earliest stages of tau aggregation that precedes, and likely facilitates, fibril formation and growth. PMID:26712030

The capture and arrangement of surface-associated microbes is influenced by biochemical and physical properties of the substrate. In this report, we develop lectin-functionalized substrates containing patterned, three-dimensional polymeric structures of varied shapes and densities and use these to investigate the effects of topology and spatial confinement on lectin-mediated microbe capture. Films of poly(glycidyl methacrylate)-block-4,4-dimethyl-2-vinylazlactone (PGMA-b-PVDMA) were patterned on silicon surfaces into line or square grid patterns with 5 m wide features and varied edge spacing. The patterned films had three-dimensional geometries with 900 nm film thickness. After surface functionalization with wheat germ agglutinin, the size of Pseudomonas fluorescens aggregates captured was dependent on the pattern dimensions. Line patterns with edge spacing of 5 m or less led to the capture of individual microbes with minimal formation of aggregates, while grid patterns with the same spacing also captured individual microbes with further reduction in aggregation. Both geometries allowed for increases in aggregate size distribution with increased in edge spacing. These engineered surfaces combine spatial confinement with affinity-based microbe capture based on exopolysaccharide content to control the degree of microbe aggregation, and can also be used as a platform to investigate intercellular interactions and biofilm formation in microbial populations of controlled sizes.

Low density lipoprotein (LDL) modified by incubation with phospholipase C (PLC-LDL) aggregates in solution and is rapidly taken up and degraded by human and mouse macrophages, producing foam cells in vitro. Human, mouse, and rabbit macrophages degraded {sup 125}I-labeled PLC-LDL ({sup 125}I-PLC-LDL) more rapidly than native {sup 125}I-labeled LDL ({sup 125}I-LDL), while nonphagocytic cells such as human fibroblasts and bovine aortic endothelial cells degraded {sup 125}I-PLC-LDL more slowly than {sup 125}I-LDL. This suggested the mechanism for internalization of PLC-LDL was phagocytosis. When examined by electron microscopy, mouse peritoneal macrophages appeared to be phagocytosing PLC-LDL. The uptake and degradation of {sup 125}I-PLC-LDL by human macrophages was inhibited >80% by the monoclonal antibody C7 (IgG2b) produced by hybridoma C7, which blocks the ligand binding domain of the LDL receptor. Similarly, methylation of {sup 125}I-LDL ({sup 125}I-MeLDL) prior to treatment with phospholipase C decreased its subsequent uptake and degradation by human macrophages by >90%. The uptake and degradation of phospholipase C-modified {sup 125}I-MeLDL by macrophages could be restored by incubation of the methylated lipoprotein with apoprotein E, a ligand recognized by the LDL receptor. These results indicate that macrophages internalize PLC-LDL by LDL receptor-dependent phagocytosis.

Multiprotein adsorption of hen egg white lysozyme at a model charged ionic surface is studied using fully atomistic molecular dynamics simulations. Simulations with two, three, and five proteins, in various orientations with respect the surface, are performed over a 100 ns time scale. Mutated proteins with point mutations at the major (Arg128 and Arg125) and minor (Arg68) surface adsorption sites are also studied. The 100 ns time scale used is sufficient to observe protein translations, rotations, adsorption, and aggregation. Two competing processes of particular interest are observed, namely surface adsorption and protein–protein aggregation. At low protein concentration, the proteins first adsorb in isolation and can then reorientate on the surface to aggregate. At high concentration, the proteins aggregate in the solution and then adsorb in nonspecific ways. This work demonstrates the role of protein concentration in adsorption, indicates the residues involved in both types of interaction (protein–protein and protein–surface), and gives an insight into processes to be considered in the development of new functionalized material systems. PMID:21671567

The misfolding and aggregation of amyloid proteins has been associated with incurable diseases such as Alzheimer's or Parkinson's disease. In the specific case of Alzheimer's disease, recent studies have shown that cell toxicity is caused by soluble oligomeric forms of aggregates appearing in the early stages of aggregation, rather than by insoluble fibrils. Research on new strategies of diagnosis is imperative to detect the disease prior to the onset of clinical symptoms. Here, we propose the use of an optical method for protein aggregation dynamic studies using a Bloch surface wave based approach. A one dimension photonic crystal made of a periodic stack of silicon oxide and silicon nitride layers is used to excite a Bloch surface wave, which is sensitive to variation of the refractive index of an aqueous solution. The aim is to detect the early dynamic events of protein aggregation and fibrillogenesis of the amyloid-beta peptide Aβ42, which plays a central role in the onset of the Alzheimer's disease. The detection principle relies on the refractive index changes caused by the depletion of the Aβ42 monomer concentration during oligomerization and fibrillization. We demonstrate the efficacy of the Bloch surface wave approach by monitoring in real-time the first crucial steps of Aβ42 oligomerization.

Radioactivity can influence surface interactions, but its effects on particle aggregation kinetics have not been included in transport modeling of radioactive particles. In this research, experimental and theoretical studies have been performed to investigate the influence of radioactivity on surface charging and aggregation kinetics of radioactive particles in the atmosphere. Radioactivity-induced charging mechanisms have been investigated at the microscopic level, and heterogeneous surface potential caused by radioactivity is reported. The radioactivity-induced surface charging is highly influenced by several parameters, such as rate and type of radioactive decay. A population balance model, including interparticle forces, has been employed to study the effects of radioactivity on particle aggregation kinetics in air. It has been found that radioactivity can hinder aggregation of particles because of similar surface charging caused by the decay process. Experimental and theoretical studies provide useful insights into the understanding of transport characteristics of radioactive particles emitted from severe nuclear events, such as the recent accident of Fukushima or deliberate explosions of radiological devices.

Assembly of nanoparticles building blocks during single crystal growth is widely observed in both natural and synthetic environments. Although this form of non-classical crystallization is generally described by oriented attachment, random aggregation of building blocks leading to single crystal products is also observed, but the mechanism of crystallographic realignment is unknown. We herein reveal that random attachment during aggregation-based growth initially produces a non-oriented growth front. Subsequent evolution of the orientation is driven by the inherent surface stress applied by the disordered surface layer and results in single crystal formation via grain boundary migration. This mechanism is corroborated by measurements of orientation rate vs external stress, demonstrating a predictive relationship between the two. These findings advance our understanding of aggregation-based growth of natural minerals by nanocrystals, and suggest an approach to material synthesis that takes advantage of stress induced co-alignment.

Productivity of fungal cultures is closely linked with their morphologic development. Morphogenesis of coagulating filamentous fungi, like Aspergillus niger, starts with aggregation of conidia, also denominated as spores. Several parameters are presumed to control this event, but little is known about their mode of action. Rational process optimization requires models that mirror the underlying reaction mechanisms. An approach in this regard is suggested and supported by experimental data. Aggregation kinetics was examined for the first 15 h of cultivation under different cultivation conditions. Mechanical stress was considered as well as pH-dependent surface interaction. Deliberations were based on a two-step aggregation mechanism. The first aggregation step is only affected by the pH-value, not by the fluid dynamic conditions in the bioreactor. The second aggregation step, in contrast, depends on the pH-value as well as on agitation and aeration induced power input. For the given experimental set-up, agitation had much more influence than aeration. In addition, hyphal growth rate was determined to be the driving force for the second aggregation step.

A coupled model, which combines the Biosphere-Atmosphere Transfer Scheme (BATS) with an advanced atmospheric boundary-layer model, was used to validate hypothetical aggregation rules for BATS-specific surface cover parameters. The model was initialized and tested with observations from the Anglo-Brazilian Amazonian Climate Observational Study and used to simulate surface fluxes for rain forest and pasture mixes at a site near Manaus in Brazil. The aggregation rules are shown to estimate parameters which give area-average surface fluxes similar to those calculated with explicit representation of forest and pasture patches for a range of meteorological and surface conditions relevant to this site, but the agreement deteriorates somewhat when there are large patch-to-patch differences in soil moisture. The aggregation rules, validated as above, were then applied to remotely sensed 1 km land cover data set to obtain grid-average values of BATS vegetation parameters for 2.8 deg x 2.8 deg and 1 deg x 1 deg grids within the conterminous United States. There are significant differences in key vegetation parameters (aerodynamic roughness length, albedo, leaf area index, and stomatal resistance) when aggregate parameters are compared to parameters for the single, dominant cover within the grid. However, the surface energy fluxes calculated by stand-alone BATS with the 2-year forcing, data from the International Satellite Land Surface Climatology Project (ISLSCP) CDROM were reasonably similar using aggregate-vegetation parameters and dominant-cover parameters, but there were some significant differences, particularly in the western USA.

In the mouse, considerable evidence indicates that initial sperm binding to the zona pellucida (ZP) is mediated by ZP3. In addition, this same glycoprotein is also responsible for inducing the acrosome reaction (AR). Whereas the O-linked oligosaccharides of ZP3 appear to mediate sperm-ZP binding, the portion of ZP3 bearing AR activity has not been defined. To try to understand the bifunctional role of ZP3 (binding and AR inducing activities), we have examined the hypothesis that ZP3 aggregates sperm receptor molecules. By analogy with findings in a variety of other extracellular signal transducing systems, including receptors for growth factors and insulin, this aggregation event could initiate the cascade resulting in the AR. To test this hypothesis, we have generated monospecific polyclonal antibodies against ZP2 and against ZP3, and examined the effects of these probes on capacitated sperm incubated in the absence or presence of various ZP protein preparations. For some experiments, we have used proteolytic fragments of ZP3, a preparation known to retain specific binding, but not AR-inducing, activity. We show here that capacitated mouse sperm, incubated with ZP glycopeptides, displayed ARs when incubated subsequently with anti-ZP3 IgG; ARs did not occur when parallel sperm samples were incubated with anti-ZP2 IgG or with anti-ZP3 Fab fragments. When capacitated sperm were treated successively, with (a) ZP3 glycopeptides, (b) anti-ZP3 Fab fragments, and (c) goat anti-rabbit IgG, ARs occurred in the majority of sperm. An alternative approach to examine this hypothesis used ZP proteins obtained from tubal eggs treated previously with bioactive phorbol diester (12-O-tetradecanoyl phorbol-13-acetate [TPA]). This preparation arrests capacitated sperm in an intermediate state of the AR. We demonstrate here that these sperm can be induced to undergo a complete AR by subsequent treatment with anti-ZP3 IgG. Together, these findings are consistent with the hypothesis

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets. PMID:3584243

The mechanism of curvature generation in membranes has been studied for decades due to its important role in many cellular functions. However, it is not clear if, or how, aggregates of lipid-anchored proteins might affect the geometry and elastic property of membranes. As an initial step toward addressing this issue, we performed structural, geometrical, and stress field analyses of coarse-grained molecular dynamics trajectories of a domain-forming bilayer in which an aggregate of lipidated proteins was asymmetrically bound. The results suggest a general mechanism whereby asymmetric incorporation of lipid-modified protein aggregates curve multidomain membranes primarily by expanding the surface area of the monolayer in which the lipid anchor is inserted. PMID:24803997

A series of experiments were performed to simulate the environmental behavior and fate of graphene oxide nanoparticles (GONPs) involved in the surface environment relating to divalent cations, natural organic matter (NOM), and hydraulics. The electrokinetic properties and hydrodynamic diameters of GONPs was systematically determined to characterize GONPs stability and the results indicated Ca(2+) (Mg(2+)) significantly destabilized GONPs with high aggregate strength factors (SF) and fractal dimension (FD), whereas NOM decreased aggregate SF with lower FD and improved GONPs stability primarily because of increasing steric repulsion and electrostatic repulsion. Furthermore, the GONPs resuspension from the sand bed into overlying water with shear flow confirmed that the release would be restricted by Ca(2+) (Mg(2+)), however, enhanced by NOM. The interaction energy based on Derjaguin-Landau-Verwey-Overbeek theory verifies the aggregation and resuspension well. Overall, these experiments provide an innovative look and more details to study the behavior and fate of GONPs.

Human amylin (hA1-37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic β-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17-29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17-29 β-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17-29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17-29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17-29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17-29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (-44% of cell viability, p aggregates. These results indicate that copper(II) influences the aggregation process and hA17-29 toxicities are especially attributable to oligomeric aggregates. hA17-29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.

Hydrophobic barium sulfate aggregates were fabricated by the direction of cocoate anions. At 30 °C, when the weight ratio of sodium cocoate to BaSO4 particles was 2.0 wt.%, the active ratio of the product reached 99.43% and the contact angle was greater than 120°. This method could not only simplify the complex modification process, but reduce energy consumption. The surface morphology, chemical structure and composition of BaSO4 aggregates were characterized by SEM, XRD, and FTIR. The results indicated that the as-synthesized BaSO4 particles were almond-liked and were composed of many interconnected nanoballs and that their surfaces were affected by cocoate anions. The adsorption of cocoate anions reversed the charge and weakened the surface polarity of BaSO4 particles, driving the formation of aggregates. And cocoate anions induced a change of the BaSO4 particles surface from hydrophilic to hydrophobic by a self-assembly and transformation process. Due to the self-assembled structure and the surface hydrophobicity, when adding the hydrophobic BaSO4 into PVC, the mechanical properties of PVC composite materials were significantly improved.

A comprehensive search for stable structures in the low coverage regime (0-1 ML) and at 2 ML and 3 ML using DFT revealed several new aggregation states of water on the non-polar ZnO(101[combining macron]0) surface. Ladder-like structures consisting of half-dissociated dimers, arranged side-by-side along the polar axis, constitute the most stable aggregate at low coverages (≤1 ML) with a binding energy exceeding that of the monolayer. At coverages beyond the monolayer - a regime that has hardly been studied previously - a novel type of structure with a continuous honeycomb-like 2D network of hydrogen bonds was discovered, where each surface oxygen atom is coordinated by additional H-bonding water molecules. This flat double-monolayer has a relatively high adsorption energy, every zinc and oxygen atom is 4-fold coordinated and every hydrogen atom is engaged in a hydrogen bond. Hence this honeycomb double monolayer offers no H-bond donor or acceptor sites for further growth of the water film. At 3 ML coverage, the interface restructures forming a contact layer of half-dissociated water dimers and a liquid-like overlayer of water attached by hydrogen bonds. The structures and their adsorption energies are analysed to understand the driving forces for aggregation and dissociation of water on the surface. We apply a decomposition scheme based on a Born-Haber cycle, discussing difficulties that may occur in applying such an analysis to the adsorption of dissociated molecules and point out alternatives to circumvent the bias against severely stretched bonds. Water aggregation on the ZnO surface is favoured by direct water-water interactions including H-bonds and dipole-dipole interactions and surface- or adsorption-mediated interactions including enhanced water-surface interactions and reduced relaxations of the water molecules and surface. While dissociation of isolated adsorbed molecules is unfavourable, partial or even full dissociation is preferred for aggregates

Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and (3H)PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of (3H)PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surfacereceptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation).

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves, Pseudomonas syringae, Pantoea agglomerans, and Pseudomonas fluorescens. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of P. fluorescens A506 and P. agglomerans 299R and mixtures of P. syringae B728a and P. agglomerans 299R than in mixtures of two isogenic strains of P. agglomerans 299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a gfp-marked strain of P. agglomerans 299R and a cfp-marked strain of P. agglomerans 299R, or of P. fluorescens A506 and P. agglomerans 299R, were similar. However, the proportion of dead cells in mixed aggregates of P. syringae B728a and P. agglomerans 299R was significantly higher (13.2% +/- 8.2%) than that in monospecific aggregates of these two strains (1.6% +/- 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the P. agglomerans 299R cells located at the interface were dead, while only about 24% of the P. syringae B728a cells were dead. The relevance of our results to understanding bacterial

Platelet Endothelial AggregationReceptor 1 (PEAR1) is a newly identified membrane protein reported to be involved in multiple vascular and thrombotic processes. While most studies to date have focused on the effects of this receptor in platelets, PEAR1 is located in multiple tissues including the endothelium, where it is most highly expressed. Our first objective was to evaluate the role of PEAR1 in endothelial function by examining flow-mediated dilation of the brachial artery in 641 participants from the Heredity and Phenotype Intervention Heart Study. Our second objective was to further define the impact of PEAR1 on cardiovascular disease computationally through meta-analysis of 75,000 microarrays, yielding insights regarding PEAR1 function, and predictions of phenotypes and diseases affected by PEAR1 dysregulation. Based on the results of this meta-analysis we examined whether genetic variation in PEAR1 influences endothelial function using an ex vivo assay of endothelial cell migration. We observed a significant association between rs12041331 and flow-mediated dilation in participants of the Heredity and Phenotype Intervention Heart Study (P = 0.02). Meta-analysis results revealed that PEAR1 expression is highly correlated with several genes (e.g. ANG2, ACVRL1, ENG) and phenotypes (e.g. endothelial cell migration, angiogenesis) that are integral to endothelial function. Functional validation of these results revealed that PEAR1 rs12041331 is significantly associated with endothelial migration (P = 0.04). Our results suggest for the first time that genetic variation of PEAR1 is a significant determinant of endothelial function through pathways implicated in cardiovascular disease.

Platelet Endothelial AggregationReceptor 1 (PEAR1) is a newly identified membrane protein reported to be involved in multiple vascular and thrombotic processes. While most studies to date have focused on the effects of this receptor in platelets, PEAR1 is located in multiple tissues including the endothelium, where it is most highly expressed. Our first objective was to evaluate the role of PEAR1 in endothelial function by examining flow-mediated dilation of the brachial artery in 641 participants from the Heredity and Phenotype Intervention Heart Study. Our second objective was to further define the impact of PEAR1 on cardiovascular disease computationally through meta-analysis of 75,000 microarrays, yielding insights regarding PEAR1 function, and predictions of phenotypes and diseases affected by PEAR1 dysregulation. Based on the results of this meta-analysis we examined whether genetic variation in PEAR1 influences endothelial function using an ex vivo assay of endothelial cell migration. We observed a significant association between rs12041331 and flow-mediated dilation in participants of the Heredity and Phenotype Intervention Heart Study (P = 0.02). Meta-analysis results revealed that PEAR1 expression is highly correlated with several genes (e.g. ANG2, ACVRL1, ENG) and phenotypes (e.g. endothelial cell migration, angiogenesis) that are integral to endothelial function. Functional validation of these results revealed that PEAR1 rs12041331 is significantly associated with endothelial migration (P = 0.04). Our results suggest for the first time that genetic variation of PEAR1 is a significant determinant of endothelial function through pathways implicated in cardiovascular disease. PMID:26406321

A limitation of the amyloid hypothesis in explaining the development of neurodegenerative diseases is that the level of amyloidogenic polypeptide in vivo is below the critical concentration required to form the aggregates observed in post-mortem brains. We discovered a novel, on-surfaceaggregation pathway of amyloidogenic polypeptide that eliminates this long-standing controversy. We applied atomic force microscope (AFM) to demonstrate directly that on-surfaceaggregation takes place at a concentration at which no aggregation in solution is observed. The experiments were performed with the full-size Aβ protein (Aβ42), a decapeptide Aβ(14-23) and α-synuclein; all three systems demonstrate a dramatic preference of the on-surfaceaggregation pathway compared to the aggregation in the bulk solution. Time-lapse AFM imaging, in solution, show that over time, oligomers increase in size and number and release in solution, suggesting that assembled aggregates can serve as nuclei for aggregation in bulk solution. Computational modeling performed with the all-atom MD simulations for Aβ(14-23) peptide shows that surface interactions induce conformational transitions of the monomer, which facilitate interactions with another monomer that undergoes conformational changes stabilizing the dimer assembly. Our findings suggest that interactions of amyloidogenic polypeptides with cellular surfaces play a major role in determining disease onset. PMID:28358113

Micrometric Au structures, presenting a dendritic nano-structure, have been fabricated on a Si-based substrate. The fabrication method involves the deposition of a thin Au film on the substrate and a high-temperature annealing (1100°C) using fast heating and cooling ramps. The thermal process produces the growth, from the substrate, of Si micro-pillars whose top surfaces, covered by a crystalline Au layer, present a nanodendritic morphology. In addition to the micro-pillars, the sample surface presents a complex structural and chemical composition including Si3N4 regions due to the silicon-nitrogen intermixing during the heating stage. By studying the kinetic processes at the Au-Si interface during the thermal treatment, we describe the stages involved in the micro-pillars growth, in the dendritic morphology development, and in the Au atoms entrapment at the top of the dendritic surfaces. Finally, we present the analyses of the optical and surface enhanced Raman scattering properties of the Au dendritic aggregates. We show, in particular, that: (1) the Au dendrites aggregates act as effective scattering elements for the electromagnetic radiation in the infrared spectral region; and (2) the higher surface area due to the branched dendritic structure is responsible for the improvement in the sensitivity of the surface enhanced Raman scattering activity.

The aggregation state of type I Fc epsilon-receptors (Fc epsilon RI) on the surface of single living mast cells was investigated by resonance fluorescence energy transfer. Derivatization of Fc epsilon RI specific ligands, i.e., immunoglobulin E or Fab fragments of a Fc epsilon RI specific monoclonal antibody, with donor and acceptor fluorophores provided a means for measuring receptor clustering through energy transfer between the receptor probes. The efficiency of energy transfer between the ligands carrying distinct fluorophores was determined on single cells in a microscope by analyzing the photobleaching kinetics of the donor fluorophore in the presence and absence of receptor ligands labeled with acceptor fluorophores. To rationalize the energy transfer data, we developed a theoretical model describing the dependence of the energy transfer efficiency on the geometry of the fluorescently labeled macromolecular ligands and their aggregation state on the cell surface. To this end, the transfer process was numerically calculated first for one pair and then for an ensemble of Fc epsilon RI bound ligands on the cell surface. The model stipulates that the aggregation state of the Fc epsilon RI is governed by an attractive lipid-protein mediated interaction potential. The corresponding pair-distribution function characterizes the spatial distribution of the ensemble. Using this approach, the energy transfer efficiency of the ensemble was calculated for different degrees of receptoraggregation. Comparison of the theoretical modeling results with the experimental energy transfer data clearly suggests that the Fc epsilon RI are monovalent, randomly distributed plasma membrane proteins. The method provides a novel approach for determining the aggregation state of cell surface components. PMID:8431535

The coherent oscillations of the surface electron gas, known as surface plasmons, in metal nanostructures can give rise to the localization of intense electromagnetic fields at the metal-dielectric interface. These strong fields are exploited in surface enhanced spectroscopies, such as Surface Enhanced Raman Scattering (SERS), for the detection and characterization of molecules at very low concentration. Still, the implementation of SERS-based biosensors requires a high level of reproducibility, combined with cheap and simple fabrication methods. For this purpose, SERS substrates based on self-assembled aggregates of commercial metallic nanoparticles (Nps) can meet all the above requests. Following this line, we report on a combined micro-Raman and Atomic Force Microscopy (AFM) analysis of the SERS efficiency of micrometric silver Np aggregates (enhancement factors up to 10{sup 9}) obtained by self-assembly. Despite the intrinsic disordered nature of these Np clusters, we were able to sort out some general rules relating the specific aggregate morphology to its plasmonic response. We found strong evidences of cooperative effects among the NPs within the cluster and namely a clear dependence of the SERS-efficiency on both the cluster area (basically linear) and the number of stacked NPs layers. A cooperative action among the superimposed layers has been proved also by electromagnetic simulations performed on simplified nanostructures consisting of stacking planes of ordered Nps. Being clear the potentialities of these disordered self-assembled clusters, in terms of both easy fabrication and signal enhancement, we developed a specific nanofabrication protocol, based on electron beam lithography and molecular functionalization, that allowed for a fine control of the Np assemblies into designed shapes fixing their area and height. In particular, we fabricated 2D ordered arrays of disordered clusters choosing gold Nps owing to their high stability. AFM measurements

The coherent oscillations of the surface electron gas, known as surface plasmons, in metal nanostructures can give rise to the localization of intense electromagnetic fields at the metal-dielectric interface. These strong fields are exploited in surface enhanced spectroscopies, such as Surface Enhanced Raman Scattering (SERS), for the detection and characterization of molecules at very low concentration. Still, the implementation of SERS-based biosensors requires a high level of reproducibility, combined with cheap and simple fabrication methods. For this purpose, SERS substrates based on self-assembled aggregates of commercial metallic nanoparticles (Nps) can meet all the above requests. Following this line, we report on a combined micro-Raman and Atomic Force Microscopy (AFM) analysis of the SERS efficiency of micrometric silver Np aggregates (enhancement factors up to 109) obtained by self-assembly. Despite the intrinsic disordered nature of these Np clusters, we were able to sort out some general rules relating the specific aggregate morphology to its plasmonic response. We found strong evidences of cooperative effects among the NPs within the cluster and namely a clear dependence of the SERS-efficiency on both the cluster area (basically linear) and the number of stacked NPs layers. A cooperative action among the superimposed layers has been proved also by electromagnetic simulations performed on simplified nanostructures consisting of stacking planes of ordered Nps. Being clear the potentialities of these disordered self-assembled clusters, in terms of both easy fabrication and signal enhancement, we developed a specific nanofabrication protocol, based on electron beam lithography and molecular functionalization, that allowed for a fine control of the Np assemblies into designed shapes fixing their area and height. In particular, we fabricated 2D ordered arrays of disordered clusters choosing gold Nps owing to their high stability. AFM measurements confirmed

Super-resolution microscopy offers unique tools for visualizing and resolving cellular structures at the molecular level. STED microscopy is a purely optical method where neither complex sample preparation nor mathematical post-processing is required. Here we present the use of STED microscopy for imaging receptor cluster composition. We use two-color STED to further determine the distribution of two different receptor subunits of the family of receptor serine/threonine kinases in the presence or absence of their ligands. The implications of receptor clustering on the downstream signaling are discussed, and future challenges are also presented.

We proposed a pancake-shaped nano-aggregate that highly focuses surface plasmons. The structure is a superposition of bowtie-shaped dimers, where surface plasmons are excited, resonated with the structure, and coupled. Surface integral equation method (Poggio-Miller-Chang-Harrington-Wu-Tsai method) is used to predict the performance of the proposed structure. It is a method which can accurately calculate the near-fields of nanoparticles. Based on the numerical prediction, the proposed structure shows an electric field (E-field) enhancement of more than 400 times, which is equivalent to a Raman enhancement factor of more than 2.5e10 times. It is promising for single molecule detections using surface-enhanced Raman scattering. The physics of the proposed structure are revealed. It is useful to design nanostructures for high E-field enhancement.

Whey proteins are now far more than a by-product of cheese processing. In the last 2 decades, food manufacturers have developed them as ingredients, with the dairy industry remaining as a major user. For many applications, whey proteins are modified (denatured) to alter their structure and functional properties. The objective of this research was to study the influence of 85 to 100 °C, with protein concentration of 8% to 12%, and treatment times of 5 to 30 min, while measuring rheological properties (storage modulus, loss modulus, and complex viscosity) and aggregation (intermolecular beta-sheet formation) in dispersions of whey protein concentrate (WPC). A Box-Behnken Response Surface Methodology modeled the heat denaturation of liquid sweet WPC at 3 variables and 3 levels. The model revealed a very significant fit for viscoelastic properties, and a lesser fit for protein aggregation, at temperatures not previously studied. An exponential increase of rheological parameters was governed by protein concentration and temperature, while a modest linear relationship of aggregation was governed by temperature. Models such as these can serve as valuable guides to the ingredient and dairy industries to develop target products, as whey is a major ingredient in many functional foods.

Scaling relationships are needed as measurements and desired predictions are often not available at concurrent spatial support volumes or temporal discretizations. Surface soil moisture values of interest to hydrologic studies are estimated using ground based measurement techniques or utilizing remote sensing platforms. Remote sensing based techniques estimate field-scale surface soil moisture values, but are unable to provide the local-scale soil moisture information that is obtained from local measurements. Further, obtaining field-scale surface moisture values using ground-based measurements is exhaustive and time consuming. To bridge this scale mismatch, we develop analytical expressions for surface soil moisture based on sharp-front approximation of the Richards equation and assumed log-normal distribution of the spatial surface saturated hydraulic conductivity field. Analytical expressions for field-scale evolution of surface soil moisture to rainfall events are utilized to obtain aggregated and disaggregated response of surface soil moisture evolution with knowledge of the saturated hydraulic conductivity. The utility of the analytical model is demonstrated through numerical experiments involving 3-D simulations of soil moisture and Monte-Carlo simulations for 1-D renderings-with soil moisture dynamics being represented by the Richards equation in each instance. Results show that the analytical expressions developed here show promise for a principled way of scaling surface soil moisture.

Gold nanoparticles (AuNP) can interact with a wide range of molecules including proteins. Whereas significant attention has focused on modifying the nanoparticle surface to regulate protein-AuNP assembly or influence the formation of the protein "corona," modification of the protein surface as a mechanism to modulate protein-AuNP interaction has been less explored. Here, we examine this possibility utilizing three small globular proteins—lysozyme with high isoelectric point (pI) and established interactions with AuNP; α-lactalbumin with similar tertiary fold to lysozyme but low pI; and myoglobin with a different globular fold and an intermediate pI. We first chemically modified these proteins to alter their charged surface functionalities, and thereby shift protein pI, and then applied multiple methods to assess protein-AuNP assembly. At pH values lower than the anticipated pI of the modified protein, AuNP exposure elicits changes in the optical absorbance of the protein-NP solutions and other properties due to aggregate formation. Above the expected pI, however, protein-AuNP interaction is minimal, and both components remain isolated, presumably because both species are negatively charged. These data demonstrate that protein modification provides a powerful tool for modulating whether nanoparticle-protein interactions result in material aggregation. The results also underscore that naturally occurring protein modifications found in vivo may be critical in defining nanoparticle-protein corona compositions.

Soil aggregates with different localization of radionuclides—(a) only on the aggregatesurface, (b) only in the intraped space, and (c) uniformly distributed throughout the aggregate volume—have been obtained under laboratory conditions, which has allowed separately assessing the roles of different aggregate parts in the uptake of radionuclides by plant roots and the reaggregation rate of the soil material. The uptake rate of the radionuclides localized on the surface of soil aggregates, especially 137Cs, by plants manifold exceeds their uptake observed at the localization of pollutants throughout the aggregate volume or only in their intraped material. The input rate of radionuclides into plants decreases with time. For 137Cs, this decrease is due to the strengthening of the sorption fixation of the radionuclide (about 15%) and the reaggregation of the soil material (85%). Under natural conditions, at a depth of 10 cm in the dark gray forest soil of a forest belt, aggregates 7-10 mm in size are subjected to 40-75% destruction and reaggregation on the average within a year, which corresponds, with consideration for the statistical dispersion of the initial data, to the aggregate lifetime of 1.5 to 3 years.

Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surfacereceptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surfacereceptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2 + signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases. PMID:23994464

α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson’s disease through the processes involved in the formation of amyloid fibrils. α and β-synuclein are homologous proteins found at comparable levels in presynaptic terminals but β-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and β-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that β-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that β-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.

α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson’s disease through the processes involved in the formation of amyloid fibrils. α and β-synuclein are homologous proteins found at comparable levels in presynaptic terminals but β-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and β-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that β-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that β-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates. PMID:27808107

Purpose: Irradiated arteries of cancer patients can be associated with atherosclerosis-like lesions containing cholesterol-laden macrophages (foam cells). Endothelial cell damage by irradiation does not completely explain the foam cell formation. We investigated the possible underlying mechanisms for ionizing radiation (IR)-induced foam cell formation. Methods and Materials: Human peripheral blood monocytes were activated by macrophage colony-stimulating factor and then treated with varying doses of IR in vitro in the absence of endothelial cells. Scavenger receptor expression and foam cell formation of IR-treated macrophages were investigated in the presence or absence of oxidized low-density lipoprotein. We also assessed the importance of mitogen-activated protein kinase activity in the macrophage colony-stimulating factor-activated human monocytes (macrophages) for the foam cell formation. Results: We found that IR treatment of macrophage colony-stimulating factor-activated human peripheral blood monocytes resulted in the enhanced expression of CD36 scavenger receptors and that cholesterol accumulated in the irradiated macrophages with resultant foam cell formation in the presence of oxidized low-density lipoprotein. Furthermore, when cultured on collagen gels, human macrophages formed large foam cell aggregates in response to IR. Antibodies against CD36 inhibited the IR-induced foam cell formation and aggregation, indicating that the IR-induced foam cell formation and the subsequent aggregation are dependent on functional CD36. In addition, we found that IR of human macrophages resulted in c-Jun N-terminal kinase activation and that c-Jun N-terminal kinase inhibition suppressed IR-induced CD36 expression and the subsequent foam cell formation and aggregation. Conclusion: Taken together, these results suggest that IR-induced foam cell formation is mediated by c-Jun N-terminal kinase-dependent CD36 activation.

For successful aqueous-based applications, it is necessary to fundamentally understand and control nanoparticle dispersivity and stability over a range of dynamic conditions, including variable ionic strengths/types, redox chemistries, and surface ligand reactivity/degradation states (i.e., surface aging). Here, we quantitatively describe the behavior of artificially aged, oleic acid (OA) bilayer coated iron oxide nanoparticles (IONPs) under different scenarios. Hydrogen peroxide (H2O2), used here as a model oxidant under both dark and light ultraviolet (UVA) conditions, was employed to "age" materials, to varying degrees, without increasing ionic strength. Short-term stability experiments indicate that OA-IONPs, while stable in the dark, are effectively destabilized when exposed to UVA/H2O2/•OH based oxidation processes. Compared to bicarbonate, phosphate (1.0 mM) has a net stabilizing effect on OA-IONPs under oxidative conditions, which can be attributed to (surface-based) functional adsorption. Corresponding aggregation kinetics in the presence of monovalent (Na(+)) and divalent cations (Ca(2+)) show that attachment efficiencies (α) are strongly dependent on the cation concentrations/types and degree of surface aging. Taken together, our findings directly highlight the need to understand the critical role of particle surface transformation(s), via oxidative aging, among other routes, with regard to the ultimate stability and environmental fate of surface functionalized engineered nanoparticles.

We have measured adsorption isotherms for two different adsorbates, neon and tetrafluoromethane, on dahlia-like carbon nanohorn aggregates. The experiments were performed at similar relative temperatures for both gases. The measurements were conducted to explore the effect of adsorbate diameter on the behavior of the resulting adsorbed systems. We measured the effective specific surface area value of the nanohorn sample using both gases, and we found that this quantity was about 22% smaller when we determined this quantity using tetrafluoromethane, the larger molecule. Isosteric heat and binding energy values were also determined from our measurements. We will compare our experimental results with those from a computer simulation study performed by Prof. M. Calbi. The simulations help us understand the source of the observed differences in the measured specific surface values, as well as the coverage dependence of the isosteric heat of adsorption for both gases.

Part of the 1994 Industrial Minerals Review. The production, consumption, and applications of construction aggregates are reviewed. In 1994, the production of construction aggregates, which includes crushed stone and construction sand and gravel combined, increased 7.7 percent to 2.14 Gt compared with the previous year. These record production levels are mostly a result of funding for highway construction work provided by the Intermodal Surface Transportation Efficiency Act of 1991. Demand is expected to increase for construction aggregates in 1995.

Hormone-dependent aggregation of the androgen receptor (AR) with a polyglutamine (polyQ) stretch amplification (>38) is considered to be the causative agent of the neurodegenerative disorder spinal and bulbar muscular atrophy (SBMA), consistent with related neurodegenerative diseases involving polyQ-extended proteins. In spite of the widespread acceptance of this common causal hypothesis, little attention has been paid to its apparent incompatibility with the observation of AR aggregation in healthy individuals with no polyQ stretch amplification. Here we used atomic force microscopy (AFM) to characterize sub-micrometer scale aggregates of the wild-type (22 glutamines) and the SBMA form (65 glutamines), as well as a polyQ deletion mutant (1 glutamine) and a variant with a normal length polyQ stretch but with a serine to alanine double mutation elsewhere in the protein. We used a baculovirus-insect cell expression system to produce full-length proteins for these structural analyses. We related the AFM findings to cytotoxicity as measured by expression of the receptors in Drosophila motoneurons or in neuronal cells in culture. We found that the pathogenic AR mutants formed oligomeric fibrils up to 300-600nm in length. These were clearly different from annular oligomers 120-180nm in diameter formed by the nonpathogenic receptors. We could also show that melatonin, which is known to ameliorate the pathological phenotype in the fly model, caused polyQ-extended AR to form annular oligomers. Further comparative investigation of these reproducibly distinct toxic and non-toxic oligomers could advance our understanding of the molecular basis of the polyQ pathologies.

Xenopus laevis melanophores express a high density (B(max) 1224 fmol/mg protein) of high-affinity (K(d) 37 pm) cell membrane melatonin receptors. Treatment of melanophores with melatonin resulted in a loss of membrane melatonin receptors reaching a maximum (approximately 60%) by 6 h. In addition to receptor loss, a decline in the potency of melatonin to produce pigment aggregation was observed on prolonged treatment. However, the loss of potency (3.8-fold in 24 h and 162-fold in 96 h) was much slower than loss of receptors, and was completely prevented by inclusion of eserine (100 microm), an inhibitor of melatonin deacetylation in the culture medium. Incubation of melanophores with [(3)H]-melatonin showed that eserine prevented metabolism of melatonin to 5-methoxytryptamine. These results indicate that although receptor density does decline on prolonged treatment, this is not responsible for the diminishing melatonin potency, which is entirely due to degradation of melatonin by deacetylation and subsequent deamination in melanophores.

Variations in receptor number at a given synapse are known to contribute to synaptic plasticity, but methods used to establish this idea usually do not allow for the determination of the dynamics of these phenomena. We used single-particle tracking to follow in real time, on the cell surface, movements of the glycine receptor (GlyR) with or without the GlyR stabilizing protein gephyrin. GlyR alternated within seconds between diffusive and confined states. In the absence of gephyrin, GlyR were mostly freely diffusing. Gephyrin induced long confinement periods spatially associated with submembranous clusters of gephyrin. However, even when most receptors were stabilized, they still frequently made transitions through the diffusive state. These data show that receptor number in a cluster results from a dynamic equilibrium between the pools of stabilized and freely mobile receptors. Modification of this equilibrium could be involved in regulation of the number of receptors at synapses.

Interactions with volcanic gases in eruption plumes produce soluble salt deposits on the surface of volcanic ash. While it has been postulated that saturation-driven precipitation of salts following the dissolution of ash surfaces by condensed acidic liquids is a primary mechanism of salt formation during an eruption, it is only recently that this mechanism has been subjected to detailed study. Here we spray water and HCl droplets into a suspension of salt-doped synthetic glass or volcanic ash particles, and produce aggregates. Deposition of acidic liquid droplets on ash particles promotes dissolution of existing salts and leaches cations from the underlying material surface. The flow of liquid, due to capillary forces, will be directed to particle-particle contact points where subsequent precipitation of salts will cement the aggregate. Our data suggest that volcanically-relevant loads of surface salts can be produced by acid condensation in eruptive settings. Several minor and trace elements mobilised by surface dissolution are biologically relevant; geographic areas with aggregation-mediated ash fallout could be “hotspots” for the post-deposition release of these elements. The role of liquids in re-distributing surface salts and cementing ash aggregates also offers further insight into the mechanisms which preserve well-structured aggregates in some ash deposits. PMID:28361966

Almost 20 years have elapsed since a phenomenon called "radial specific coalescence" was identified. During studies of electrolytic oxygen evolution from the back side of a vertically oriented, transparent tin oxide electrode in alkaline electrolyte, one of the authors (Sides) observed that large "collector" bubbles appeared to attract smaller bubbles. The bubbles moved parallel to the surface of the electrode, while the electric field was normal to the electrode surface. The phenomenon was reported but not explained. More recently self ordering of latex particles was observed during electrophoretic deposition at low DC voltages likewise on a transparent tin oxide electrode. As in the bubble work, the field was normal to the electrode while the particles moved parallel to it. Fluid convection caused by surface induced flows (SIF) can explain these two apparently different experimental observations: the aggregation of particles on an electrode during electrophoretic deposition, and a radial bubble coalescence pattern on an electrode during electrolytic gas evolution. An externally imposed driving force (the gradient of electrical potential or temperature), interacting with the surface of particles or bubbles very near a planar conducting surface, drives the convection of fluid that causes particles and bubbles to approach each other on the electrode.

Benzodiazepines are among the most prescribed compounds and are commonly present in many toxicological screens. They are also of concern forensically in cases of drug facilitated sexual assault. Currently these compounds are predominantly analyzed using immunoassay techniques; however more specific screening methods are needed. This paper demonstrates the applicability of surface enhanced Raman spectroscopy as a method for the analysis and detection of benzodiazepines. The procedure involves mixing urine extracts with gold nanoparticles and appropriate aggregating agents for trace detection of these compounds and their metabolites. In this paper we will discuss the optimization of various parameters of this technique as well as its application to screening urine samples. Eleven different benzodiazepines and metabolites were examined, including 1,2-triazolo-benzodiazepines and 1,4-benzodiazpines. Experiments were performed using four different chloride salts, MgCl2, CaCl2, KCl, and NaCl, as aggregating agents for the colloidal gold nanoparticles. Overall it was found that each aggregating agent produced different levels of signal enhancement for each drug. MgCl2 provided the lowest limit of detection at 2.5 ng mL(-1), and linearity over a wide range of concentrations for a variety of drugs chosen. It was also determined that the optimum MgCl2 concentration was 1.67 M. This method has shown the applicability of SERS for the detection of trace quantities of benzodiazepines in aqueous solutions as well as the optimization of the technique over a wide range of compounds. This technique can be utilized in the detection of trace benzodiazepines in toxicological samples following extraction of the analyte.

Lightweight Expanded Clay Aggregate (LECA) modified with an aqueous solution of magnesium chloride MgCl2 and hydrogen peroxide H2O2 was used to remove Cr(VI) from aqueous solutions. The adsorption properties of the used adsorbents were investigated through batch studies, Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), X-ray Fluorescence Spectroscopy (XRF), and Fourier Transform Infrared (FTIR) spectroscopy. The effect created by magnesium chloride on the modification of the LECA surface was greater than that of hydrogen peroxide solution and showed a substantial increase in the specific surface area which has a value of 76.12 m2/g for magnesium chloride modified LECA while the values of 53.72 m2/g, and 11.53 m2/g were found for hydrogen peroxide modified LECA and natural LECA, respectively. The extent of surface modification with enhanced porosity in modified LECA was apparent from the recorded SEM patterns. XRD and FTIR studies of themodified LECA surface did not show any structural distortion. The adsorption kinetics was found to follow the modified Freundlich kinetic model and the equilibrium data fitted the Sips and Dubinin-Radushkevich equations better than other models. Maximum sorption capacities were found to be 198.39, 218.29 and 236.24 mg/g for natural LECA, surface modified LECA with H2O2 and surface modified LECA with MgCl2, respectively. Adsorbents were found to have only a weak effect on conductivity and turbidity of aqueous solutions. Spent natural and surface modified LECA with MgCl2 was best regenerated with HCl solution, while LECA surface modified with H2O2 was best regenerated with HNO3 concentrated solution. Thermal method showed a lower regeneration percentage for all spent adsorbents.

The adsorption and self-assembly of surfactants are ubiquitous processes in several technological applications, including the manufacture of nano-structured materials using bottom-up strategies. Although much is known about the adsorption of surfactants on homogeneous flat surfaces from experiments, theory, and simulations, limited information is available, in quantifiable terms, regarding the adsorption of surfactants on surfaces with chemical and/or morphological heterogeneity. In an effort to fill this knowledge gap, we report here results obtained using equilibrium dissipative particle dynamics (DPD) simulations for the adsorption of model surfactants onto patterned flat surfaces (i.e., flat surfaces with chemical heterogeneity). The patterns consist of one or two stripes of variable width on which the surfactants could adsorb. The adsorbing stripes are surrounded by a surface that effectively repels the surfactants. This repelling surface, perhaps not realistic, allows us to quantify the effect of lateral confinement on the morphology of surfactant aggregates. When the stripe width is large (effectively providing a homogeneous flat surface), the surfactants yield a flat monolayer. Our simulations suggest that the flat monolayers become hemi-cylinders, hemi-spheres, and individual surfactants as the stripe width decreases, a consequence of lateral confinement. In some cases our simulations show evidence of cooperative effects when two adsorbing stripes are present on the surface. If the distance between the stripes and the widths of the stripes are both less than about one surfactant length, hemi-cylindrical shells and irregular structures are observed because of cooperativity; otherwise the results match those found for a single isolated stripe. Our predictions could be useful for the design of new nano-structured materials and coatings, for applications ranging from nano-fluidic devices to nano-reactors.

Most-probable-number (MPN) dilution series were used to enumerate and isolate bacteria from bulk water, suspended aggregates, the oxic layer, and the oxic-anoxic transition zone of the sediment of a tidal flat ecosystem in the southern North Sea. The heterotrophic aerobic bacteria were able to grow on agar-agar, alginate, cellulose, chitin, dried and ground Fucus vesiculosus, Marine Broth 2216, palmitate, and starch. MPN counts of bulk water and aggregate samples ranged between 0.18 × 101 and 1.1 × 106 cells per milliliter and those of the sediment surface and the transition zone between 0.8 × 101 and 5.1 × 107 cells per gram dry weight. Marine Broth and F. vesiculosus yielded the highest values of all substrates tested and corresponded to 2.3-32% of 4,6-diamidinophenyl indole cell counts. Strains of seven phylogenetic classes were obtained: Actinobacteria, Bacilli, α- and γ-Proteobacteria, Sphingobacteria, Flavobacteria, and Planctomycetacia. Only with agar-agar as substrate could organisms of all seven classes be isolated.

Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor–receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional “trapping,” and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain α7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the α7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain. PMID

The development of a novel methodology for extraction and preconcentration of the most commonly used anionic surface active agents (SAAs), linear alkylbenzene sulfonates (LAS), is presented herein. The present method, based on the use of silica-magnetite nanoparticles modified with cationic surfactant aggregates, was developed for determination of C10-C13 LAS homologues. The proposed methodology allowed quantitative recoveries of C10-C13 LAS homologues by using a reduced amount of magnetic nanoparticles. Limits of detection were in the range 0.8-1.9μgL(-1) for C10-C13 LAS homologues, while the repeatability, expressed as relative standard deviation (RSD), ranged from 2.0 to 3.9% (N=6). Finally, the proposed method was successfully applied to the analysis of a variety of natural water samples.

Testing of P2Y12-receptor antagonist effects can support clinical decision-making. However, most platelet function assays use only ADP as agonist which is not P2Y12-receptor specific. For this reason P2Y12-receptor specific assays have been developed by adding prostaglandin E1 (PGE1) to reduce ADP-induced platelet activation via the P2Y1-receptor. The present study sought to evaluate a P2Y12-receptor specific assay for determination of pharmacodynamic and clinical outcomes. This study enrolled 400 patients undergoing coronary stenting after loading with clopidogrel or prasugrel. ADP-induced platelet reactivity was assessed by whole blood aggregometry at multiple time points with a standard ADP assay (ADPtest) and a P2Y12-receptor specific assay (ADPtest HS, both run on Multiplate Analyzer, Roche Diagnostics). Patients were clinically followed for 1 month and all events adjudicated by an independent committee. In total, 2084 pairs of test results of ADPtest and ADPtest HS were available showing a strong correlation between results of both assays (r = 0.96, p < 0.001). These findings prevailed in multiple prespecified subgroups (e.g., age; body mass index; diabetes). Calculated cutoffs for ADPtest HS and the established cutoffs of ADPtest showed a substantial agreement for prediction of ischemic and hemorrhagic events with a Cohen's κ of 0.66 and 0.66, respectively. The P2Y12-receptor specific ADPtest HS assay appears similarly predictive for pharmacodynamic and clinical outcomes as compared to the established ADPtest assay indicating its applicability for clinical use. Further evaluation in large cohorts is needed to determine if P2Y12-receptor specific testing offers any advantage for prediction of clinical outcome.

In this article we report the synthesis and in vitro evaluation of well-defined, folate functionalized and fluorescently labeled polymers based on the clinically approved N-(2-hydroxypropyl)-methacrylamide (HPMA). The polymers were prepared applying the RAFT polymerization method as well as the reactive ester approach. The molecular weights of the polymers synthesized were around 15 and 30 kDa. The total content of conjugated folate varied from 0, 5, and 10 mol %. The cellular uptake of these polymers was investigated in the folate receptor (FR)-positive human nasopharyngeal epidermal carcinoma (KB-3-1) and FR-negative human lung epithelial carcinoma (A549) cancer cell lines. In FR-positive cells, the cellular uptake of polymers depended strongly on the folate content. The conjugates with the highest folate content led to the highest level of cell-associated fluorescence. Regarding influence of molecular weight, nonsignificant differences were observed when total cell uptake was analyzed. The cellular uptake is related to the aggregate formation of the polymer conjugates, which were studied by fluorescence correlation spectroscopy (FCS). For the conjugates, we found aggregates with a diameter ranging from 11-18 nm. Much to our surprise, we found aggregates of the same size for the 30 kDa polymer bearing 5 mol % folate and for the 15 and 30 kDa conjugates with a folate content of 10 mol %. Consequently, a different conformation in solution for the different conjugates was expected. By live cell confocal fluorescence microscopy the receptor-mediated endocytosis process was observed, as colocalization with lysosomal markers was achieved. In addition, cellular uptake was not observed in FR-negative cells (A549) and can be dramatically reduced by blocking the FR with free folic acid. Our findings clearly underline the need for a minimum amount of accessible folate units to target the FR that triggers specific cellular uptake. Furthermore, it has been demonstrated that

Red Blood Cell (RBC) aggregation is an important hemorheological phenomenon especially in microcirculation. In healthy individuals, RBCs are known to aggregate and gravitate toward the faster flow in the center of vessels to increase their throughput for more efficient oxygen delivery. Their aggregation is known to occur during a variety of environmental, pathological, and physiological conditions and is reversible when aggregates are subject to the relatively high shear forces in the circulation. The likelihood that aggregates will monodisperse in flow is dependent on the conditions during which they form. In situations where such aggregates are not sheared to monodispersion their presence can impact the perfusion of microvascular networks. More specifically, aggregates subject to the low shear rates in the zone of stasis near regions of thermal burn injury are capable of occluding vessels in the microcirculation and inhibiting the delivery of oxygen and nutrients to tissue downstream. The basic mechanism leading to erythrocyte aggregation at the onset of thermal injury is unknown. This dissertation investigates parameters involved in erythrocyte aggregation, methods of measuring and testing erythrocyte aggregation, and incorporates modeling based on first principles ultimately to propose a mechanism of this phenomenon.

The plasmonic resonances of nanostructured silver films produce exceptional surface enhancement, enabling reproducible single-molecule Raman scattering measurements. Supporting a broad range of plasmonic resonances, these disordered systems are difficult to investigate with conventional far-field spectroscopy. Here, we use nonlinear excitation spectroscopy and polarization anisotropy of single optical hot spots of supercontinuum generation to track the transformation of these plasmon modes as the mesoscopic structure is tuned from a film of discrete nanoparticles to a semicontinuous layer of aggregated particles. We demonstrate how hot spot formation from diffractively-coupled nanoparticles with broad spectral resonances transitions to that from spatially delocalized surface plasmon excitations, exhibiting multiple excitation resonances as narrow as 13 meV. Photon-localization microscopy reveals that the delocalized plasmons are capable of focusing multiple narrow radiation bands over a broadband range to the same spatial region within 6 nm, underscoring the existence of novel plasmonic nanoresonators embedded in highly disordered systems. PMID:23807624

Thin films of SnO{sub 2}, nanocrystallites have been surface-modified with thionine, methylene blue, and oxazine 170 by adsorption from the corresponding dye solutions. The strong electrostatic interaction between the cationic dye and the negatively charged semiconductor nanocrystallites results in close packing of the dye on the semiconductor surface. These closely packed H-aggregates of the adsorbed dye are active both electrochemically and photoelectrochemically. Electron transfer from semiconductor nanocrystallites into the adsorbed dye aggregates leads to bleaching of the colored film. The extent of dye bleaching which is readily controlled by the applied potential, has been probed by spectroelectrochemical measurements. The photocurrent action spectra of these dye-modified SnO{sub 2} films indicate charge injection from excited dye aggregate into the semiconductor nanocrystallites with an incident photon-to-photocurrent efficiency of < 1 %.

new class of chemical therapeutics for treatment of prostate cancer. 15. SUBJECT TERMS X-ray crystallography, high throughput screening, medicinal... treatments because anti-androgen resis- tance usually develops. We conducted functional and x-ray screens to identify compounds that bind the AR surface and...possibility that such compounds could be used for prostate cancer treatment . It is unlikely that natural T3 or Triac concentrations approach levels required

The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the internalization of Y5R upon stimulation with the TrkB ligand, BDNF. Upon treatment with BDNF, the cells were exposed to a membrane impermeable, biotinylation reagent that selectively labels surface proteins. Subsequently, the biotinylated membrane proteins were affinity-purified on columns with avidin resins and analyzed by Western blot. Differences in the fraction of receptors present on the cell surface of control and ligand-treated cells served as a measure of their internalization and response to particular stimuli.

Posttranslational protein modifications can play a major role in disease pathogenesis; phosphorylation, sumoylation, and acetylation modulate the toxicity of a variety of proteotoxic proteins. The androgen receptor (AR) is substantially modified, in response to hormone binding, by phosphorylation, sumoylation, and acetylation; these modifications might thus contribute to DHT-dependent polyglutamine (polyQ)-expanded AR proteotoxicity in spinal and bulbar muscular atrophy (SBMA). SIRT1, a nuclear protein and deacetylase of the AR, is neuroprotective in many neurodegenerative disease models. Our studies reveal that SIRT1 also offers protection against polyQ-expanded AR by deacetylating the AR at lysines 630/632/633. This finding suggested that nuclear AR acetylation plays a role in the aberrant metabolism and toxicity of polyQ-expanded AR. Subsequent studies revealed that the polyQ-expanded AR is hyperacetylated and that pharmacologic reduction of acetylation reduces mutant AR aggregation. Moreover, genetic mutation to inhibit polyQ-expanded AR acetylation of lysines 630/632/633 substantially decreased its aggregation and completely abrogated its toxicity in cell lines and motor neurons. Our studies also reveal one means by which the AR acetylation state likely modifies polyQ-expanded AR metabolism and toxicity, through its effect on DHT-dependent AR stabilization. Overall, our findings reveal a neuroprotective function of SIRT1 that operates through its deacetylation of polyQ-expanded AR and highlight the potential of both SIRT1 and AR acetylation as powerful therapeutic targets in SBMA.

Dynamic behaviors of cell aggregates on a dendrimer surface were investigated to drive the directed differentiation of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic lineage. Cell aggregates on the polyamidoamine dendrimer surface with fifth-generation (G5) of dendron structure showed dynamic changes in morphology associated with repetitive stretching and contracting during migration. Spatial-temporal observations revealed cellular movement in single aggregates by their morphological change through stretching and contracting on the G5 surface, suggesting that the dynamic behavior of aggregate causes mixing of cells. However, aggregates without cell-substrate adhesions on the low-binding culture surface sustained their spherical morphology without cellular movement within a single aggregate. Furthermore, β-catenin was observed at nuclei in aggregates on the G5 surface, and expression of the cardiomyocyte marker cardiac Troponin T (cTnT) was detected. However, β-catenin localized to the nuclei only in the outer region of the aggregate on the low-binding culture surface, and cTnT expression was restricted at the exterior surface of the aggregates. These observations indicate that cell mixing within aggregates on the G5 surface induced the directed differentiation of hMSCs toward a cardiomyogenic lineage by nuclear translocation of β-catenin through dissociation of cell-cell adhesions. These results suggest that migration-driven aggregate behaviors on the dendrimer surface caused repeated morphological changes of aggregate through stretching and contracting, leading to the directed differentiation of hMSCs toward a cardiomyogenic fate commitment.

Enzyme immobilization has been of great industrial importance because of its use in various applications like bio-fuel cells, bio-sensors, drug delivery and bio-catalytic films. Although research on enzyme immobilization dates back to the 1970's, it has been only in the past decade that scientists have started to address the problems involved systematically. Most of the previous works on enzyme immobilization have been retrospective in nature i.e enzymes were immobilized on widely used substrates without a compatibility study between the enzyme and the substrate. Consequently, most of the enzymes lost their activity upon immobilization onto these substrates due to many governing factors like protein-surface and inter-protein interactions. These interactions also play a major role biologically in cell signaling, cell adhesion and inter-protein interactions specifically is believed to be the major cause for neurodegenerative diseases like Alzheimer's and Parkinson's disease. Therefore understanding the role of these forces on proteins is the need of the hour. In my current research, I have mainly focused on two factors a) Surface Curvature b) Surface Chemistry as both of these play a pivotal role in influencing the activity of the enzymes upon immobilization. I study the effect of these factors computationally using a stochastic method known as Monte Carlo simulations. My research work carried out in the frame work of a Hydrophobic-Polar (HP) lattice model for the protein shows that immobilizing enzymes inside moderately hydrophilic or hydrophobic pores results in an enhancement of the enzymatic activity compared to that in the bulk. Our results also indicate that there is an optimal value of surface curvature and hydrophobicity/hydrophilicity where this enhancement of enzymatic activity is highest. Further, our results also show that immobilization of enzymes inside hydrophobic pores of optimal sizes are most effective in mitigating protein-aggregation. These

The wetting and evaporative aggregation of alumina nanofluids (Al2O3) are examined for CVD-synthesized graphene-coated (GC) surfaces that are known as strongly hydrophobic (θcontact ≈ 90°). Our findings are compared to those associated with a hydrophilic cover glass (CG) substrate (θcontact ≈ 45°). The nanofluidic self-assemblies on the GC substrate are elaborately characterized in terms of the droplet wetting/crack formation, the particle migration time over the evaporative time (CR), the Derjaguin-Landau-Verwey-Overbeek forces (FDLVO), and the relative thermal conductivity (KR). The GC substrate forms relatively thicker and larger cracks and requires a longer evaporation time. Both the GC and CG substrates share approximately the same time constant CR, which suggests the formation of coffee-ring patterns for both substrates. The GC shows negative FDLVO, which implies a repulsive force between the nanoparticles and the substrate, and the CG shows a positive FDLVO of attraction. Furthermore, a more than 3 order of magnitude larger thermal conductivity of GC compared to that of CG drives significantly different particle/fluid motions near the drop edge areas between the two substrates.

Temporally composited remote sensing products are widely used in monitoring the urban heat island (UHI). In order to quantify the impact of temporal aggregation for assessing the UHI, we examined MODIS land surface temperature (LST) products for 11 years focusing on Houston, Texas and its surroundings. By using the daily LST from 2000 to 2010, the urban and rural daily LST were presented for the 8-day period and annual comparisons for both day and night. Statistics based on the rural-urban LST differences show that the 8-day composite mean UHI effects are generally more intensive than that calculated by daily UHI images. Moreover, the seasonal pattern shows that the summer daytime UHI has the largest magnitude and variation while nighttime UHI magnitudes are much smaller and less variable. Regression analyses enhance the results showing an apparently higher UHI derived from 8-day composite dataset. The summer mean UHI maps were compared, indicating a land cover related pattern. We introduced yearly MODIS land cover type product to explore the spatial differences caused by temporal aggression of LST product. The mean bias caused by land cover types are calculated about 0.5 ~ 0.7K during the daytime, and less than 0.1K at night. The potential causes of the higher UHI are discussed. The analysis shows that the land-atmosphere interactions, which result in the regional cloud formation, are the primary reason.

AFRL-AFOSR-UK-TR-2011-0002 First Principles Calculations on the Diffusion of Cu, Ag and Au Atoms or Aggregates on the Bulk and...SUBTITLE First Principles Calculations on the Diffusion of Cu, Ag and Au Atoms or Aggregates on the Bulk and Surface of Titania 5a. CONTRACT...093072 Final report First principles calculations on the diffusion of Cu, Ag and Au atoms or aggregates on the bulk and surface of titania List

G protein-coupled receptors (GPCRs) mediate physiological responses to a diverse array of stimuli and are the molecular targets for numerous therapeutic drugs. GPCRs primarily signal from the plasma membrane, but when expressed in heterologous cells many GPCRs exhibit poor trafficking to the cell surface. Multiple approaches have been taken to enhance GPCR surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones. In addition to allowing for enhanced surface expression of certain GPCRs in heterologous cells, these approaches have also shed light on the control of GPCR trafficking in vivo and in some cases have led to new therapeutic approaches for treating human diseases that result from defects in GPCR trafficking. PMID:19679364

Research highlights: {yields} Vitamin D deficiency is linked to accelerated decline in lung function. {yields} Levels of vitamin D receptor (VDR) are decreased in lungs of patients with COPD. {yields} VDR knock-out mouse showed increased lung inflammation and emphysema. {yields} This was associated with decline in lung function and increased MMPs. {yields} VDR knock-out mouse model is useful for studying the mechanisms of lung diseases. -- Abstract: Deficiency of vitamin D is associated with accelerated decline in lung function. Vitamin D is a ligand for nuclear hormone vitamin D receptor (VDR), and upon binding it modulates various cellular functions. The level of VDR is reduced in lungs of patients with chronic obstructive pulmonary disease (COPD) which led us to hypothesize that deficiency of VDR leads to significant alterations in lung phenotype that are characteristics of COPD/emphysema associated with increased inflammatory response. We found that VDR knock-out (VDR{sup -/-}) mice had increased influx of inflammatory cells, phospho-acetylation of nuclear factor-kappaB (NF-{kappa}B) associated with increased proinflammatory mediators, and up-regulation of matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-12 in the lung. This was associated with emphysema and decline in lung function associated with lymphoid aggregates formation compared to WT mice. These findings suggest that deficiency of VDR in mouse lung can lead to an early onset of emphysema/COPD because of chronic inflammation, immune dysregulation, and lung destruction.

G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surfacereceptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we

The capacity of human neutrophils to bind PAF was rapidly diminished upon cell stimulation with both physiological agonists (N-formylmethionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4)) and pharmacologic agonists (phorbol 12-myristate 13-acetate (PMA), A23187). As a consequence, PAF responses in neutrophils were blunted, as monitored by an inhibition of intracellular Ca2+ mobilization. Downregulation of the PAF receptor in neutrophils by diverse agonists was temperature-sensitive and required intact cells. Scatchard analysis of binding data revealed that PAF binding sites were lost without an appreciable change in the affinity of the ligand for the receptor. The binding of the PAF receptor antagonist WEB2086 to neutrophils decreased in parallel with PAF binding. PMA-induced PAF receptor downregulation was staurosporine-sensitive while PAF receptor downregulation by A23187, FMLP, or LTB4 was staurosporine-resistant. Both neutrophil aggregation (a form of intercellular adhesion) and PAF receptor downregulation occurred only at high concentrations of agonists while other signaling processes such as the increase in [Ca2+]i, PKC activation, and PAF synthesis were stimulated at low concentrations of agonists. Furthermore, agonist-induced PAF receptor downregulation was observed only under conditions in which the activated neutrophils were stirred (or shaken) and were allowed to aggregate. Additionally, chelation of extracellular Ca2+ with EGTA minimized cell aggregation and also inhibited PAF receptor downregulation. While the nature of the biochemical signal or the physical changes in the plasma membrane associated with aggregation or that follow aggregation remain to be elucidated it is clear that full expression of cell activation (i.e., neutrophil aggregation) is required for PAF receptor downregulation.

A critical event in atherogenesis is the interaction of arterial wall macrophages with subendothelial lipoproteins. Although most studies have investigated this interaction by incubating cultured macrophages with monomeric lipoproteins dissolved in media, arterial wall macrophages encounter lipoproteins that are mostly bound to subendothelial extracellular matrix, and these lipoproteins are often aggregated or fused. Herein, we utilize a specialized cell-culture system to study the initial interaction of macrophages with aggregated low density lipoprotein (LDL) bound to extracellular matrix. The aggregated LDL remains extracellular for a relatively prolonged period of time and becomes lodged in invaginations in the surface of the macrophages. As expected, the degradation of the protein moiety of the LDL was very slow. Remarkably, however, hydrolysis of the cholesteryl ester (CE) moiety of the LDL was 3-7-fold higher than that of the protein moiety, in stark contrast to the situation with receptor-mediated endocytosis of acetyl-LDL. Similar results were obtained using another experimental system in which the degradation of aggregated LDL protein was delayed by LDL methylation rather than by retention on matrix. Additional experiments indicated the following properties of this interaction: (a) LDL-CE hydrolysis is catalyzed by lysosomal acid lipase; (b) neither scavenger receptors nor the LDL receptor appear necessary for the excess LDL-CE hydrolysis; and (c) LDL-CE hydrolysis in this system is resistant to cellular potassium depletion, which further distinguishes this process from receptor-mediated endocytosis. In summary, experimental systems specifically designed to mimic the in vivo interaction of arterial wall macrophages with subendothelial lipoproteins have demonstrated an initial period of prolonged cell-surface contact in which CE hydrolysis exceeds protein degradation.

Phospholipid vesicles are used as substrates to form adsorbed aggregates of 2,2'-cyanine, also referred to as pseudoisocyanine (PIC). In this paper, we report photophysical parameters of two putative adsorbed aggregates species (cis- and trans-aggregates, relating to their makeup from mono-cis and all-transstereoisomers, respectively). Phase modulation picosecond fluorescence decay measurements reveal that superradiance and energy transfer are dominant features controlling photophysical processes. Superradiance, coherence size, energy transfer and exciton-phonon coupling are discussed for the two types of aggregates; as regards photophysical parameters, the fluorescence lifetimes, fluorescence quantum yields, and nonradiative rate constants are determined. It is suggested that structure plays the crucial role in excited state dynamics.

P(2)Y(12) receptor is a G(i)-coupled adenosine diphosphate (ADP) receptor with a critical role in platelet aggregation. It contains two potential N-linked glycosylation sites at its extra cellular amino-terminus, which may modulate its activity. Studies of both tunicamycin treatment and site-directed mutagenesis have revealed a dispensable role of the N-linked glycosylation in the receptor's surface expression and ligand binding activity. However, the non-glycosylated P(2)Y(12) receptor is defective in the P(2)Y(12)-mediated inhibition of the adenylyl cyclase activity. Thus the study uncovers an unexpected vital role of N-linked glycans in receptor's signal transducing step but not in surface expression or ligand binding.

A new series of pH-regulated asymmetric amino-acid gemini surfactants N,N'-dialkyl-N,N'-diacetate ethylenediamine (Ace(m)-2-Ace(n)), differing by the asymmetric degree and length of the carbon tails (m = 8 and 10, n = 10, 12, 14, and 16), were synthesized in three steps. On the basis of pKa values obtained by pH titration, surface tension, fluorescence, dynamic light scattering (DLS), and transmission electron microscopy (TEM) measurements were performed to study the surface adsorption and aggregation properties in aqueous Ace(m)-2-Ace(n) solution. The new compounds have higher surface activity and better pH adaptability in comparison with that of symmetric gemini surfactants Ace(n)-2-Ace(n). The molecule behavior of Ace(m)-2-Ace(n) can be adjusted by either the hydrophobic group or the pH. With increasing alkyl chain length, the surface adsorption declines but its ability to form aggregates increases. We find that pH can promote the self-assembly transition of Ace(m)-2-Ace(n) from surfactant monomers to aggregates through protonation between H(+) and the tertiary nitrogen group. TEM data further confirm the pH-regulated molecular self-assembly process and the existence of vesicles at neutral or weak acidic pH. pH-recyclability is found to be reversible by pH-light transmittance recycle tests.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuron disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. Ligand-dependent nuclear accumulation of mutant AR protein is a critical characteristic of the pathogenesis of SBMA. SBMA has been modeled in AR-overexpressing animals, but precisely how the polyglutamine (polyQ) expansion leads to neurodegeneration is unclear. Induced pluripotent stem cells (iPSCs) are a new technology that can be used to model human diseases, study pathogenic mechanisms, and develop novel drugs. We established SBMA patient-derived iPSCs, investigated their cellular biochemical characteristics, and found that SBMA-iPSCs can differentiate into motor neurons. The CAG repeat numbers in the AR gene of SBMA-iPSCs and also in the atrophin-1 gene of iPSCs derived from another polyQ disease, dentato-rubro-pallido-luysian atrophy (DRPLA), remain unchanged during reprogramming, long term passage, and differentiation, indicating that polyQ disease-associated CAG repeats are stable during maintenance of iPSCs. The level of AR expression is up-regulated by neuronal differentiation and treatment with the AR ligand dihydrotestosterone. Filter retardation assays indicated that aggregation of ARs following dihydrotestosterone treatment in neurons derived from SBMA-iPSCs increases significantly compared with neurological control iPSCs, easily recapitulating the pathological feature of mutant ARs in SBMA-iPSCs. This phenomenon was not observed in iPSCs and fibroblasts, thereby showing the neuron-dominant phenotype of this disease. Furthermore, the HSP90 inhibitor 17-allylaminogeldanamycin sharply decreased the level of aggregated AR in neurons derived from SBMA-iPSCs, indicating a potential for discovery and validation of candidate drugs. We found that SBMA-iPSCs possess disease-specific biochemical features and could thus open new avenues of research into not only SBMA, but also other polyglutamine diseases.

A new prototype of a Hamilton receptor suitable for the functionalization of inorganic nanoparticles was synthesized and characterized. The hydrogen bonding receptor was coupled to a catechol moiety, which served as anchor group for the functionalization of metal oxides, in particular zinc oxide. Synthesized zinc oxide nanorods [ZnO] were used for surface functionalization. The wet-chemical functionalization procedure towards monolayer-grafted particles [ZnO-HR] is described and a detailed characterization study is presented. In addition, the detection of specific cyanurate molecules is demonstrated. The hybrid structures [ZnO-HR-CA] were stable towards agglomeration and exhibited enhanced dispersability in apolar solvents. This observation, in combination with several spectroscopic experiments gave evidence of the highly directional supramolecular recognition at the surface of nanoparticles.

The present study was performed to investigate the combined effects of the platelet glycoprotein IIb/IIIa receptor antagonist c7E3 Fab (abciximab) and the anticoagulants unfractionated heparin (UH), low molecular weight heparin (LMWH), and recombinant hirudin (rH) on platelet aggregation and thrombin generation under high coagulant challenge by extrinsic activation of platelet-rich plasma. Platelet aggregation and thrombin generation were assessed simultaneously in the presence of different concentrations of abciximab and anticoagulants. Increasing concentrations of abciximab resulted in a dose-dependent anti-aggregating effect with a maximum at 20 microg/ml. Doses of 5, 10, and 20 microg/ml abciximab prolonged the lag phase until the onset of platelet aggregation, but this effect was independent of the dosage used. Abciximab had no influence on the thrombin potential under our high coagulant challenge. UH, LMWH, and rH showed a dose-dependent prolongation of the lag phase until the onset of platelet aggregation and decreased the thrombin potential. Addition of anticoagulants did not contribute to further inhibition of platelet aggregation in the presence of abciximab, but the combination of abciximab and anticoagulants exhibited an additive effect on prolongation of the lag phase until the onset of platelet aggregation. Addition of abciximab to anticoagulants did not result in further decrease of the thrombin potential. Our study demonstrates the respective specific effects of abciximab and anticoagulants on platelet aggregation and thrombin potential under high coagulant challenge, and also an additive effect of abciximab and the anticoagulants UH, LMWH, and rH on the lag phase until the onset of platelet aggregation.

The subject of this work is the synthesis of polymer nanostructures via the use of surfactant surfaceaggregates as templates, also termed Template Assisted Admicellar Polymerization (TAAP). The first chapter reviews some of the most current nanopatterning techniques (including both top-down and bottom-up approaches), with particular emphasis on the fabrication of organic and inorganic patterned nanostructures via particle lithography. In chapter 2, highly ordered hexagonal arrays of latex spheres were prepared on highly ordered pyrolytic graphite (HOPG) from a variation of the Langmuir Blodgett technique, using an anionic surfactant (SDS), and a low molecular weight (ca. 10000) polyacrylamide as spreading agents. When a nonionic polyethoxylated (EO = 9) surfactant was used as the spreading agent, no ordered arrays were observed. Based on the correlation found between the surface tension in the presence of the latex particles and the critical concentration at which hexagonal arrangements of latex spheres occurs; a model was proposed to explain the role of the spreading agent in forming stable monolayers at the air/liquid interface, which in turn are necessary for the formation of well-ordered monolayers on a solid substrate from the LB technique. According to this model, solid-like regions of small numbers of latex spheres form at the liquid-air interface, which are then transferred to the substrate. These ordered regions then act as nuclei for the formation of 2D arrays of latex spheres on the surface upon water evaporation. The role of other factors such as relative humidity, substrate and solvent choice, and pulling vs. compression speed were also found to affect the quality of the monolayers formed. Finally, a simple, easy to automate, yet effective surface tension method was proposed to predict the optimal conditions for the formation of ordered monolayers using a variation of the LB deposition method from any monodisperse set of spheres. In chapter 3, a novel

Continuous monitoring of surface energy fluxes provides an important tool for precision agriculture management. It is, therefore, desirable to obtain these fluxes at agricultural field size (length scale ~ 10-100 m). To date, land surface temperature (LST), a fundamental input required for flux computations, is usually available at a nominal resolution of 1 km, which disables field-scale monitoring. Disaggregating LST data into field-scale sub-pixels was found to be possible, with deterioration in temperature accuracy as sub-pixel size is reduced. In contrast to LST, land use and fractional vegetation cover (LU and FC, additional key inputs) are available at high spatial resolution (e.g., 30 m). Aggregation of LU and FC to meet the lower resolution LST data introduces errors when aggregating to larger pixel sizes. The objective of this research is to find the optimum resolution that will minimize the errors due to aggregation of LU/FC and disaggregation of LST data, to provide continuous estimates of field scale surface energy fluxes. Data were used from the 2002 Soil Moisture-Atmosphere Coupling Experiment (SMACEX02) conducted over the upper Midwest corn and soybean production region of Iowa. Three dates during the period of rapid crops growth (June 23, July 1, and July 8) for which Landsat TM images are available were analyzed. The original pixels were aggregated to form 960 m pixels (to mimic thermal data currently available from MODIS) and were then disaggregated following the procedure suggested by Kustas et al. (2003)* to form 60, 120, and 240 m sub-pixels. LU and FC were obtained at 30 m resolution and then aggregated to 60, 120, 240, and 960 m. The Two-Source-Model was run at each of the resolutions using the pertinent inputs. The model output at 60 m resolution, using the original LST data was considered the base line, to which all other outputs were compared. For comparing the flux results at the lower resolutions, the 60 m flux output was aggregated. The

Humoral immunity is that aspect of specific immunity that is mediated by B lymphocytes and involves the neutralizing of pathogens by means of antibodies attaching to the pathogen's binding sites. Antibodies bind to and block ligand sites on the pathogen which prevents these sites from attaching to target cell receptors and so cell entry is inhibited. Many studies investigate the role of humoral immunity for protection against chlamydial challenge and they have shown that neutralization of the chlamydial body requires a large number of attached antibodies. Steric hindrance greatly influences the number of available sites that may be bound, reducing relative occupancy well below 100%. We model steric effects of antibody Fab fragment attachment indicating that they must be taken into consideration to accurately model valency, the number of available binding sites. We derive a partial differential equation for the number of antibody Fabs and host cell receptors that are aggregated to extracellular chlamydial elementary bodies. We consider steric effects in describing the size distribution of aggregates. Our theory is in good agreement with Monte Carlo simulations of binding. We use our theoretical prediction for the valency in a model for the in-host population dynamics of a chlamydial infection and we fit our model to experimental data.

By in situ time-lapse AFM, we investigated early-stage aggregates of PrP formed at low concentration (100 ng/mL) on mica and Au(111) surfaces in acetate buffer (pH 4.5). Remarkably different PrP assemblies were observed. Oligomeric structures of PrP aggregates were observed on mica surface, which was in sharp contrast to the multi-layer PrP aggregates yielding parallel linear patterns observed Au(111) surface. Combining molecular dynamics and docking simulations, PrP monomers, dimers and trimers were revealed as the basic units of the observed aggregates. Besides, the mechanisms of the observed PrP aggregations and the corresponding molecular-substrate and intermolecular interactions were suggested. These interactions involved gold-sulfur interaction, electrostatic interaction, hydrophobic interaction, and hydrogen binding interaction. In contrast, the PrP aggregates observed in pH 7.2 PBS buffer demonstrated similar large ball-like structures on both mica and Au(111) surfaces. The results indicate that the pH of a solution and the surface of the system can have strong effects on supramolecular assemblies of prion proteins. This study provides in-depth understanding on the structural and mechanistic nature of PrP aggregation, and can be used to study the aggregation mechanisms of other proteins with similar misfolding properties.

Extract from seeds and bark of horse chestnut (Aesculus hippocastanum L) is used as an herbal medicine against chronic venous insufficiency. The effect and mechanism of action on veins, arteries, and platelets are not fully understood. The aim of this study was to investigate the effects and mechanisms of action of horse chestnut on the contraction of bovine mesenteric veins and arteries, and human platelet aggregation. Contraction studies showed that horse chestnut extract dose-dependently contracted both veins and arteries, with the veins being the most sensitive. Contraction of both veins and arteries were significantly inhibited by the 5-HT(2A) receptor antagonist ketanserin. No effect on contraction was seen with the cyclooxygenase inhibitor indomethacin, the alpha(1) receptor antagonist prazosin or the angiotensin AT(1) receptor antagonist saralasin neither in veins nor arteries. ADP-induced human platelet aggregation was significantly reduced by horse chestnut. A further reduction was seen with the extract in the presence of ketanserin. In conclusion, horse chestnut contraction of both veins and arteries is, at least partly, mediated through 5-HT(2A) receptors. Human platelet aggregation is reduced by horse chestnut. The clinical importance of these findings concerning clinical use, possible adverse effects, and drug interactions remains to be investigated.

Quercetin and rhamnose were efficiently converted into huaicarbon A/B by heating at 250°C for 10-15 min or at 200°C for 25-30 min. With the optimum molar ratio of quercetin/rhamnose (1:3), huaicarbon A and B yields reached 25% and 16% respectively after heating at 250°C, with 55% quercetin conversion. Huaicarbon A/B both promoted washed platelet aggregation dose-dependently, which was antagonized by an inhibitor of glycoprotein VI (GPVI) receptor. Similarly, they both promoted collagen-induced platelet aggregation in platelet-rich plasma in dose-dependent manners. According to the S type dose-response model, EC50 values of huaicarbon A and huaicarbon B were calculated as 33.48 μM and 48.73 μM respectively. They induced intracellular Ca2+ accumulation that was specifically blocked by GPVI antagonist. Huaicarbon A/B enhanced intracellular Ca2+ accumulation and facilitated collagen-induced platelet aggregation, which were blocked by GPVI antagonist. They were conducive to collagen-induced platelet aggregation by activating platelet GPVI receptor. PMID:28337278

Based on a contiguous and structurally as well as biologically diverse set of 87 sigma(1) ligands, a 5D-QSAR study was conducted in which a quasi-atomistic receptorsurface modeling approach (program package Quasar) was applied. The superposition of the ligands was performed with the tool Pharmacophore Elucidation (MOE-package), which takes all conformations of the ligands into account. This procedure led to four pharmacophoric structural elements with aromatic, hydrophobic, cationic and H-bond acceptor properties. Using the aligned structures a 3D-model of the ligand binding site of the sigma(1) receptor was obtained, whose general features are in good agreement with previous assumptions on the receptor structure, but revealed some novel insights since it represents the receptorsurface in more detail. Thus, e.g., our model indicates the presence of an H-bond acceptor moiety in the binding site as counterpart to the ligands' cationic ammonium center, rather than a negatively charged carboxylate group. The presented QSAR model is statistically valid and represents the biological data of all tested compounds, including a test set of 21 ligands not used in the modeling process, with very good to excellent accuracy [q(2) (training set, n=66; leave 1/3 out) = 0.84, p(2) (test set, n=21)=0.64]. Moreover, the binding affinities of 13 further spirocyclic sigma(1) ligands were predicted with reasonable accuracy (mean deviation in pK(i) approximately 0.8). Thus, in addition to novel insights into the requirements for binding of spirocyclic piperidines to the sigma(1) receptor, the presented model can be used successfully in the rational design of new sigma(1) ligands.

Alzheimer's disease (AD) is a multifactorial syndrome with several target proteins contributing to its etiology. To confront AD, an innovative strategy is to design single chemical entities able to simultaneously modulate more than one target. Here, we present compounds that inhibit acetylcholinesterase and NMDA receptor activity. Furthermore, these compounds inhibit AChE-induced Abeta aggregation and display antioxidant properties, emerging as lead candidates for treating AD.

1. PGD2 (DP)-receptors mediate inhibition of platelet aggregation and vasodilatation. If receptor reserve were greater on platelets it might be possible to separate these effects. To determine whether such a difference in receptor reserve exists, we have examined the effects of a highly selective DP-receptor partial agonist 192C86 on platelet aggregation and the cardiovascular system in healthy volunteers. 2. Using an open, dose-escalating study design, four male volunteers received constant rate intravenous infusions of 192C86 for up to 60 min. Ex vivo platelet aggregation to ADP and collagen in platelet-rich plasma (PRP) and whole blood (WB) was studied at baseline, after 15, 30 and 60 min of each infusion and at 180 min post-infusion. Heart rate (HR), systolic and diastolic (DBP) blood pressure were measured at frequent intervals. Adverse experiences were monitored by checklist. Facial flushing was assessed by the volunteer using a visual analogue scale, by an observer using a numerical scale and by full-face colour photographs. Blood was taken for assay of plasma 192C86 concentrations by radio-immunoassay (r.i.a.). 3. 192C86 (0.007-0.058 micrograms kg-1 min-1) inhibited platelet aggregation to ADP and collagen both in PRP and WB in a dose-dependent manner. However, this was always accompanied by a decrease in DBP, increase in HR and facial flushing. Plasma concentrations of 192C86 were at or below the limits of sensitivity of the r.i.a. (0.5 ng ml-1). 4. The highest infusion rate was stopped after 20 min due to symptomatic hypotension on standing.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human interleukin-1 receptor antagonist (IL-1ra) in aqueous solutions unfolds and aggregates when subjected to hydrostatic pressures greater than about 180 MPa. This study examined the mechanism and thermodynamics of pressure-induced unfolding and aggregation of IL-1ra. The activation free energy for growth of aggregates (ΔG∓aggregation) was found to be 37 ± 3 kJ/mol, whereas the activation volume (ΔV∓aggregation) was −120 ± 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, ΔGdenaturation, was 20 ± 5 kJ/mol, whereas the partial specific volume change for denaturation, ΔVdenaturation, was −110 ± 30 mL/mol. When IL-1ra begins to denature at pressures near 140 MPa, cysteines that are normally buried in the native state become exposed. Under oxidizing conditions, this results in the formation of covalently cross-linked aggregates containing nonnative, intermolecular disulfide bonds. The apparent activation free energy for nucleation of aggregates, ΔG∓nuc, was 42 ± 4 kJ/mol, and the activation volume for nucleation, ΔV∓nuc,was −175 ± 37 mL/mol, suggesting that a highly solvent-exposed conformation is needed for nucleation. We hypothesize that the large specific volume of IL-1ra, 0.752 ± 0.004 mL/g, coupled with its relatively low conformational stability, leads to its susceptibility to denaturation at relatively low pressures. The positive partial specific adiabatic compressibility of IL-1ra, 4.5 ± 0.7 ± 10−12 cm2/dyn, suggests that a significant component of the ΔVdenaturation is attributable to the elimination of solvent-free cavities. Lastly, we propose that hydrostatic pressure is a useful variable to conduct accelerated formulation studies of therapeutic proteins. PMID:16081653

The relationship between the nature of micro damage under impact loading and changes in mechanical behavior associated with different microstructures is studied for concretes made with two different coarse aggregates having significant differences mainly in roughness and porosity - sintered fly ash and uncrushed gravel. A range of techniques including X-ray diffraction, digital image analysis, mercury porosimetry, X-ray computed tomography, laser surface profilometry and scanning electron microscopy were used to characterize the aggregates and micro-structures. The concrete prepared with lightweight aggregates was stronger in compression than the gravel aggregate concrete due to enhanced hydration as a result of internal curing. In the lightweight concrete, it was deduced that an inhomogeneous micro-structure led to strain incompatibilities and consequent localized stress concentrations in the mix, leading to accelerated failure. The pore structure, compressibility, and surface texture of the aggregates are of paramount importance for the micro-cracking growth.

CD44 is a broadly distributed cell surface protein thought to mediate cell attachment to extracelular matrix components or specific cell surface ligands. We have created soluble CD44-immunoglobulin fusion proteins and characterized their reactivity with tissue sections and lymph node high endothelial cells in primary culture. The CD44 target on high endothelial cells is sensitive to enzymes that degrade hyaluronate, and binding of soluble CD44 is blocked by low concentrations of hyaluronate or high concentrations of chondroitin 4- and 6-sulfates. A mouse anti-hamster hyaluonate receptor antibody reacts with COS cells expressing hamster CD44 cDNA. In sections of all tissues examined, including lymph nodes and Peyer's patches, predigestion with hyaluronidase eliminated CD44 binding.

α-Crystallin is a multimeric protein belonging to the family of small heat shock proteins, which function as molecular chaperones by resisting heat and oxidative stress induced aggregation of other proteins. We immobilized α-Crystallin on a self-assembled monolayer on glass surface and studied its activity in terms of the prevention of aggregation of aldolase. We discovered that playing with grafted protein density led to interesting variations in the chaperone activity of immobilized α-Crystallin. This result is in accordance with the hypothesis that dynamicity of subunits plays a vital role in the functioning of α-Crystallin and might be able to throw light on the structure-activity relationship. We showed that the chaperone activity of a certain number of immobilized α-Crystallins was superior compared to a solution containing an equivalent number of the protein and 10 times the number of the protein at temperatures >60 °C. The α-Crystallin grafted surfaces retained activity on reuse. This could also lead to the design of potent heat-shock resistant surfaces that can find wide applications in storage and shipping of protein based biopharmaceuticals.

Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet-granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet-granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood.

Abstract We present the mapping of the full plasmonic mode spectrum for single and aggregated gold nanoparticles linked through DNA strands to a silicon nitride substrate. A comprehensive analysis of the electron energy loss spectroscopy images maps was performed on nanoparticles standing alone, dimers, and clusters of nanoparticles. The experimental results were confirmed by numerical calculations using the Mie theory and Gans-Mie theory for solving Maxwell's equations. Both bright and dark surface plasmon modes have been unveiled. PACS 78.67.Bf; 61.46.Df; 87.64.Ee PMID:23890222

Natural killer (NK) cells play a major role in innate defenses against pathogens, primarily viruses, and are also thought to be part of the immunosurveillance against tumors. They express an array of surfacereceptors that mediate NK cell function. The human leukocytes antigen (HLA) class I-specific inhibitory receptors allow NK cells to detect and kill cells that have lost or under-express HLA class I antigens, a typical feature of tumor or virally infected cells. However, NK cell activation and induction of cytolytic activity and cytokine production depends on another important checkpoint, namely the expression on target cells of ligands recognized by activating NK receptors. Despite their potent cytolytic activity, NK cells frequently fail to eliminate tumors. This is due to mechanisms of tumor escape, determined by the tumor cells themselves or by tumor-associated cells (i.e. the tumor microenvironment) via the release of soluble suppressive factors or the induction of inhibitory loops involving induction of regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells. The most important clinical application involving NK cells is the cure of high-risk leukemias in the haplo-identical hematopoietic stem cell transplant (HSCT) setting. NK cells originated from hematopoietic stem cells (HSC) of HLA-haploidentical donors may express Killer Immunoglobulin-like receptors (KIRs) that are mismatched with the HLA class I alleles of the recipient. This allows NK cells to kill leukemia blasts residual after the conditioning regimen, while sparing normal cells (that do not express ligands for activating NK receptors). More recent approaches based on the specific removal of TCR α/β(+) T cells and of CD19(+) B cells, allow the infusion, together with CD34(+) HSC, of mature KIR(+) NK cells and of TCR γ/δ(+) T cells, both characterized by a potent anti-leukemia activity. This greatly reduces the time interval necessary to obtain alloreactive, KIR(+) NK

Strong coupling regime is reached when SPPs and matter exchange energy coherently and reversibly before losses take place, resulting in the formation of new hybrid exciton-plasmon states formed by lower and upper energy bands [1,2]. In this field most of the works focus on steady-state observations, indeed experiments on the dynamics of such hybrid systems are relatively scarce and their intrinsic photophysics is still far from being understood. Here, in order to improve our understanding of the dynamics of hybrid exciton-plasmon states, we have studied through ultrafast pump-probe approach, a hybrid system composed by gold hole arrays and J-aggregate molecules while modifying the lattice constant of the metallic array. Under upper hybrid band resonant excitation, transient absorption spectra provide the evidence that exciton-plasmon hybrid states are formed. Meanwhile, kinetics analysis led to the discovery of a remarkably long-lived upper band, at least one order of magnitude at 1/e than bare J-aggregate molecules. This result was explained with the identification, in the transient absorption spectra, of a trap state combined with the negligible relaxation effect from vibrational modes. The intrinsic long lifetime of hybrid states is of crucial importance both from a fundamental and applicative point of view, having implications in the use of exciton-plasmon states for technological purposes. The understanding of the dynamics on strong coupling systems can provide indeed a promising route towards novel ultrafast plasmonic devices with coherent functionalities.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis. PMID:21454471

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys(6), Lys(27), and Lys(29) linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys(6), Lys(27), and Lys(29) ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surfacereceptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes.

The NASA Thermal Infrared Multispectral Scanner (TIMS) has been successfully used for the remote identification of a variety of soil and aggregate deposits in vegetated areas of two states. Over three million cubic meters of gravel deposits were identified from the imagery during a two year period. Verification was accomplished by ground reconnaissance using drilling machinery and by ground instrumentation. The method has been used to differentiate between fine and coarse grained soils, and gravel deposits. The deposits were found to have been naturally sorted according to grain size by depositional processes, providing each deposit with distinct spectral qualities. It was found that the masking effects of relatively dense vegetation were largely overcome by using imagery acquired at higher altitudes above terrain than 9000 meters, due to loss of resolution of the finer detail. The mechanics of image resolution are discussed, a method of data analysis used is described, and sample spectral signatures are illustrated.

Strongly activated "coated" platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbβ3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein "coat." Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as "cap," a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbβ3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather "capped") platelets to become incorporated into thrombi despite their lack of active integrins.

The evaporation of a tiny amount of water on the solid surface with different wettabilities has been studied by molecular dynamics simulations. From nonequilibrium MD simulations, we found that, as the surface changed from hydrophobic to hydrophilic, the evaporation speed did not show a monotonic decrease as intuitively expected, but increased first, and then decreased after it reached a maximum value. The analysis of the simulation trajectory and calculation of the surface water interaction illustrate that the competition between the number of water molecules on the water-gas surface from where the water molecules can evaporate and the potential barrier to prevent those water molecules from evaporating results in the unexpected behavior of the evaporation. This finding is helpful in understanding the evaporation on biological surfaces, designing artificial surfaces of ultrafast water evaporating, or preserving water in soil.

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surfaceaggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, and is used to remove them and to reveal the underlying active layer. The systematic analysis of the surfaceaggregates suggests that the structure consists of PCBM molecules. PMID:28382247

A derivative of the hypothalamic peptide luteinizing hormone-releasing hormone (LHRH) has been coupled to ferritin and the conjugate purified by gel chromatography. In its ability to stimulate the secretion of luteinizing hormone from pituitary cells in vitro, the conjugate has the same potency and specificity as the native peptide. When dissociated pituitary cells maintained in short-term culture are lightly fixed with formaldehyde and then incubated with the conjugate, examination in the electron microscope shows an even distribution of ferritin particles over the free cell surface of the gonadotrophin cells. This binding appears to be specific for the LHRH receptor since it is prevented by a 10-fold excess of native peptide. In addition to the gonadotrophin cells, some somatotrophin and thyrotrophin cells bind conjugate on their free surfaces under similar conditions. If living cells are incubated with the conjugate for 15 min, the bound conjugate becomes aggregated and then concentrated in one localized area of the cell surface. In this area, which lies immediately above the juxtanuclear Golgi complex, the plasma membrane is frequently invaginated in a manner which suggests that the bound, aggregated conjugate is internalized by endocytosis. PMID:233747

Recurrent airway obstruction (RAO) is an allergic disease of horses similar to human asthma, which is characterized by airway inflammation and activation of neutrophils, lymphocytes and platelets. Platelet activation and an increase in circulating platelet-leukocyte aggregates may lead to airway remodeling. The aim of this study was to investigate platelet status in RAO-affected horses based on the platelet morphology and platelet surface expression of CD41/61 and CD62P. Ten RAO-affected horses and ten healthy horses were included in this study. Blood samples were obtained to determine the platelet count (PLT), mean platelet volume (MPV) and platelet large cell ratio (P-LCR). Expression of CD62P and CD41/61 was detected by flow cytometry on activated platelets. The median PLT was significantly reduced in horses with RAO compared to the controls. The MPV and the P-LCR values were significantly higher in RAO horses than controls. Expression of CD41/61 on platelets was increased in RAO horses, while CD62P expression was reduced. This study demonstrated the morphological changes in platelets and expression of platelet surfacereceptors. Despite the decrease of CD62P expression, the observed increased surface expression of CD41/61 on platelets in horses with RAO may contribute to the formation of platelet aggregates in their respiratory system.

Aggregation of cell surfacereceptors by multivalent ligand can trigger a variety of cellular responses. A well-studied receptor that responds to aggregation is the high affinity receptor for IgE (FcepsilonRI), which is responsible for initiating allergic reactions. To quantify antigen-induced aggregation of IgE-FcepsilonRI complexes, we have developed a method based on multiparameter flow cytometry to monitor both occupancy of surface IgE combining sites and association of antigen with the cell surface. The number of bound IgE combining sites in excess of the number of bound antigens, the number of bridges between receptors, provides a quantitative measure of IgE-FcepsilonRI aggregation. We demonstrate our method by using it to study the equilibrium binding of a haptenated fluorescent protein, 2,4-dinitrophenol-coupled B-phycoerythrin (DNP25-PE), to fluorescein isothiocyanate-labeled anti-DNP IgE on the surface of rat basophilic leukemia cells. The results, which we analyze with the aid of a mathematical model, indicate how IgE-FcepsilonRI aggregation depends on the total concentrations of DNP25-PE and surface IgE. As expected, we find that maximal aggregation occurs at an optimal antigen concentration. We also find that aggregation varies qualitatively with the total concentration of surface IgE as predicted by an earlier theoretical analysis. PMID:10233059

To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 {+-} 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.

We report here a detailed structural and magnetic study of different silica nanocapsules containing uniform and highly crystalline maghemite nanoparticles. The magnetic phase consists of 5 nm triethylene glycol (TREG)- or dimercaptosuccinic acid (DMSA)-coated maghemite particles. TREG-coated nanoparticles were synthesized by thermal decomposition. In a second step, TREG ligands were exchanged by DMSA. After the ligand exchange, the ζ potential of the particles changed from - 10 to - 40 mV, whereas the hydrodynamic size remained constant at around 15 nm. Particles coated by TREG and DMSA were encapsulated in silica following a sol-gel procedure. The encapsulation of TREG-coated nanoparticles led to large magnetic aggregates, which were embedded in coalesced silica structures. However, DMSA-coated nanoparticles led to small magnetic clusters inserted in silica spheres of around 100 nm. The final nanostructures can be described as the result of several competing factors at play. Magnetic measurements indicate that in the TREG-coated nanoparticles the interparticle magnetic interaction scenario has not dramatically changed after the silica encapsulation, whereas in the DMSA-coated nanoparticles, the magnetic interactions were screened due to the function of the silica template. Moreover, the analysis of the AC susceptibility suggests that our systems essentially behave as cluster spin glass systems.

Soil cover and soil management are the factors that most influence soil erosion by water, because they directly affect soil surface roughness and surface cover. The main effect of soil cover by crop residues consists in dissipation of kinetic energy of raindrops and also partly kinetic energy of runoff, so that the soil disaggregation is considerably reduced but, in addition, soil cover captures detached soil particles, retains water on its surface and decreases runoff volume and velocity. In turn, soil surface roughness, influences soil surface water storage and infiltration and also runoff volume and velocity, sediment retention and subsequently water and sediment losses. Based on the above rationale, we performed a field experiment to assess the influence of soil cover and soil surface roughness on decay of runoff velocity as well as on mean diameter of transported sediments (D50 index). The following treatments were evaluated: SRR) residues of Italian ryegrass (Lolium multiflorum) on a smooth soil surfcace, SRV) residues of common vetch (Vicia sativa) on a smooth soil surface, SSR) scarification after cultivation of Italian ryegrass resulting in a rough surface, SSV) scarification after cultivation of common vetch resulting in a rough surface, and SBS) scarified bare soil with high roughness as a control. The field experiments was performed on an Inceptisol in South Brazil under simulated rainfall conditions during 2012. Experimental plots were 11 m long and 3.5 m wide with an area of 38.5 m2. Six successive simulated rainfall tests were applied using a rotating-boom rain simulator. During each test, rain intensity was 60 mmhr-1, whereas rain duration was 90 minutes. Runoff velocity showed no significant differences between cultivated treatments. However, when compared to bare soil treatment, SBS (0.178 m s-1) and irrespective of the presence of surface crop residues or scarification operations, cultivated soil treatments significantly reduced runoff velocity

Nanosized drug carriers functionalized with moieties specifically targeting tumor cells are promising tools in cancer therapy, due to their ability to circulate in the bloodstream for longer periods and their selectivity for tumor cells, enabling the sparing of healthy tissues. Because of its biocompatibility, high bioresorbability, and responsiveness to pH changes, synthetic biomimetic nanocrystalline apatites are used as nanocarriers to produce multifunctional nanoparticles, by coupling them with the chemotherapeutic drug doxorubicin (DOXO) and the DO-24 monoclonal antibody (mAb) directed against the Met/Hepatocyte Growth Factor receptor (Met/HGFR), which is over-expressed on different types of carcinomas and thus represents a useful tumor target. The chemical-physical features of the nanoparticles are fully investigated and their interaction with cells expressing (GTL-16 gastric carcinoma line) or not expressing (NIH-3T3 fibroblasts) the Met/HGFR is analyzed. Functionalized nanoparticles specifically bind to and are internalized in cells expressing the receptor (GTL-16) but not in the ones that do not express it (NIH-3T3). Moreover they discharge DOXO in the targeted GTL-16 cells that reach the nucleus and display cytotoxicity as assessed in an MTT assay. Two different types of ternary nanoparticles are prepared, differing for the sequence of the functionalization steps (adsorption of DOXO first and then mAb or vice versa), and it is found that the ones in which mAb is adsorbed first are more efficient under all the examined aspects (binding, internalization, cytotoxicity), possibly because of a better mAb orientation on the nanoparticle surface. These multifunctional nanoparticles could thus be useful instruments for targeted local or systemic drug delivery, allowing a reduction in the therapeutic dose of the drug and thus adverse side effects. Moreover, this work opens new perspectives in the use of nanocrystalline apatites as a new platform for theranostic

The interactions of human genotype AO erythrocytes (red blood cells) (RBCs) with N-acetylgalactosamine-reactive lectins isolated from Helix pomatia (HPA) and from Dolichos biflorus (DBA) were studied. Binding curves obtained with the use of tritium-labeled lectins showed that the maximal numbers of lectin molecules capable of binding to human genotype AO RBCs were 3.8 X 10(5) and 2.7 X 10(5) molecules/RBC for HPA and DBA, respectively. The binding of one type of lectin may influence the binding of another type. HPA was found to inhibit the binding of DBA, but not vice versa. The binding of HPA was weakly inhibited by a beta-D-galactose-reactive lectin isolated from Ricinus communis (designated RCA1). Limulus polyphemus lectin (LPA), with specificity for N-acetylneuraminic acid, did not influence the binding of HPA but enhanced the binding of DBA. About 80% of LPA receptors (N-acetylneuraminic acid) were removed from RBC surfaces by neuraminidase treatment. Neuraminidase treatment of RBCs resulted in increases of binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 X 10(5) of new HPA sites were generated on genotypes AO and OO RBCs by neuraminidase treatment, and these new sites accounted for the enhancement (AO cells) and appearance (OO cells) of hemagglutinability by HPA. Neuraminidase treatment did not generate new DBA sites, but increased the DBA affinity for the existing receptors; as a result, genotype AO cells increased their hemagglutinability by DBA, while OO cells remained unagglutinable. The use of RBCs of different genotypes in binding assays with 3H-labeled lectins of known specificities provides an experimental system for studying cell-cell recognition and association.

In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer.

The authors have studied the binding of 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein (fluorescein biotin) to 6.2 {micro}m diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. They have determined the average number of binding sites per bead, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. The authors estimate the site number to be {approx}1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is {approx}(1.3 {+-} 0.3) {times} 10{sup 7} 1{sup {minus}1} S{sup {minus}1}. The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation rate constant is as high as 0.05 s{sup {minus}1} shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.

The ability to adhere to salivary agglutinin-coated hydroxyapatite beads and to aggregate in the presence of fluid-phase salivary agglutinin was tested by using 25 isolates of mutants streptococci representing eight serotypes. Both adherence and aggregation activity correlated with expression of the Mr-185,000 cell surface antigen P1 on Streptococcus mutans serotype c, e, and f strains. In addition, it was shown that the P1 molecule itself served as the adhesin of S. mutans serotype c, since adherence was significantly inhibited by the presence of recombinant-specified Mr-150,000 P1. The ability of S. sobrinus strains to adhere or aggregate did not correlate with expression of the P1 cross-reactive antigen SpaA. There was also evidence for interaction with salivary agglutinin, as manifested by aggregation but not adherence of S. rattus serotype b, which does not express a P1 cross-reactive antigen. To understand the interaction of P1 with salivary agglutinin at the molecular level, a panel of 11 anti-P1 monoclonal antibodies was tested for inhibitory activity in adherence and aggregation inhibition assays. Overlapping, but not identical, subsets of monoclonal antibodies were found to inhibit adherence and aggregation, indicating that the interactions of P1 with salivary agglutinin which mediate these two phenomena are different. The localization of functional domains of P1 which may mediate the aggregation and adherence reactions is discussed. PMID:1541515

The self-assembly of lipid bilayer membranes to enclose functional biomolecules, thus defining a “protocell,” was a seminal moment in the emergence of life on Earth and likely occurred at the micro-environment of the mineral-water interface. Mineral-lipid interactions are also relevant in biomedical, industrial and technological processes. Yet, no structure-activity relationships (SARs) have been identified to predict lipid self-assembly at mineral surfaces. Here we examined the influence of minerals on the self-assembly and survival of vesicles composed of single chain amphiphiles as model protocell membranes. The apparent critical vesicle concentration (CVC) increased in the presence of positively-charged nanoparticulate minerals at high loadings (mg/mL) suggesting unfavorable membrane self-assembly in such situations. Above the CVC, initial vesicle formation rates were faster in the presence of minerals. Rates were correlated with the mineral’s isoelectric point (IEP) and reactive surface area. The IEP depends on the crystal structure, chemical composition and surface hydration. Thus, membrane self-assembly showed rational dependence on fundamental mineral properties. Once formed, membrane permeability (integrity) was unaffected by minerals. Suggesting that, protocells could have survived on rock surfaces. These SARs may help predict the formation and survival of protocell membranes on early Earth and other rocky planets, and amphiphile-mineral interactions in diverse other phenomena. PMID:28266537

The self-assembly of lipid bilayer membranes to enclose functional biomolecules, thus defining a "protocell," was a seminal moment in the emergence of life on Earth and likely occurred at the micro-environment of the mineral-water interface. Mineral-lipid interactions are also relevant in biomedical, industrial and technological processes. Yet, no structure-activity relationships (SARs) have been identified to predict lipid self-assembly at mineral surfaces. Here we examined the influence of minerals on the self-assembly and survival of vesicles composed of single chain amphiphiles as model protocell membranes. The apparent critical vesicle concentration (CVC) increased in the presence of positively-charged nanoparticulate minerals at high loadings (mg/mL) suggesting unfavorable membrane self-assembly in such situations. Above the CVC, initial vesicle formation rates were faster in the presence of minerals. Rates were correlated with the mineral's isoelectric point (IEP) and reactive surface area. The IEP depends on the crystal structure, chemical composition and surface hydration. Thus, membrane self-assembly showed rational dependence on fundamental mineral properties. Once formed, membrane permeability (integrity) was unaffected by minerals. Suggesting that, protocells could have survived on rock surfaces. These SARs may help predict the formation and survival of protocell membranes on early Earth and other rocky planets, and amphiphile-mineral interactions in diverse other phenomena.

The self-assembly of lipid bilayer membranes to enclose functional biomolecules, thus defining a “protocell,” was a seminal moment in the emergence of life on Earth and likely occurred at the micro-environment of the mineral-water interface. Mineral-lipid interactions are also relevant in biomedical, industrial and technological processes. Yet, no structure-activity relationships (SARs) have been identified to predict lipid self-assembly at mineral surfaces. Here we examined the influence of minerals on the self-assembly and survival of vesicles composed of single chain amphiphiles as model protocell membranes. The apparent critical vesicle concentration (CVC) increased in the presence of positively-charged nanoparticulate minerals at high loadings (mg/mL) suggesting unfavorable membrane self-assembly in such situations. Above the CVC, initial vesicle formation rates were faster in the presence of minerals. Rates were correlated with the mineral’s isoelectric point (IEP) and reactive surface area. The IEP depends on the crystal structure, chemical composition and surface hydration. Thus, membrane self-assembly showed rational dependence on fundamental mineral properties. Once formed, membrane permeability (integrity) was unaffected by minerals. Suggesting that, protocells could have survived on rock surfaces. These SARs may help predict the formation and survival of protocell membranes on early Earth and other rocky planets, and amphiphile-mineral interactions in diverse other phenomena.

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.

Colony stimulating factor-1 receptor (CSF-1R), a receptor tyrosine kinase (RTK), is the master regulator of macrophage biology. CSF-1 can bind CSF-1R resulting in receptor activation and signalling essential for macrophage functions such as proliferation, differentiation, survival, polarization, phagocytosis, cytokine secretion, and motility. CSF-1R activation can only occur after the receptor is presented on the macrophage cell surface. This process is reliant upon the underlying macrophage receptor trafficking machinery. However, the mechanistic details governing this process are incompletely understood. C-terminal Eps15 Homology Domain-containing (EHD) proteins have recently emerged as key regulators of receptor trafficking but have not yet been studied in the context of macrophage CSF-1R signalling. In this manuscript, we utilize primary bone-marrow derived macrophages (BMDMs) to reveal a novel function of EHD1 as a regulator of CSF-1R abundance on the cell surface. We report that EHD1-knockout (EHD1-KO) macrophages cell surface and total CSF-1R levels are significantly decreased. The decline in CSF-1R levels corresponds with reduced downstream macrophage functions such as cell proliferation, migration, and spreading. In EHD1-KO macrophages, transport of newly synthesized CSF-1R to the macrophage cell surface was reduced and was associated with the shunting of the receptor to the lysosome, which resulted in receptor degradation. These findings reveal a novel and functionally important role for EHD1 in governing CSF-1R signalling via regulation of anterograde transport of CSF-1R to the macrophage cell surface.

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.

Rabbit alveolar macrophages which were treated at 0 degrees C with phenylarsine oxide and then incubated at 37 degrees C for 10 min exhibited a two- to threefold increase in surfacereceptor activity for macroglobulin.protease complexes, diferric transferrin, and mannose- terminal glycoproteins. Analysis of the concentration-dependence of ligand binding indicated that changes in ligand-binding activity were due to changes in receptor number rather than alterations in ligand- receptor affinity. Surfacereceptor number could also be increased by treatment of cells with three other sulfhydryl reagents, N- ethylmaleimide, p-chloromercurobenzoate, and iodoacetic acid. The increase in receptor activity was maximal after 10 min and decreased over the next hour. This decrease in cell-associated receptor activity was due to the release of large membrane vesicles which demonstrated a uniform buoyant density by isopycnic sucrose gradient centrifugation. Treatment of cells with phenylarsine oxide did not decrease the cellular content of lactate dehydrogenase or beta-galactosidase, indicating that cell integrity was maintained and lysosomal enzyme release did not occur. Our studies indicate that phenylarsine oxide treatment in the presence of extracellular Ca2+ results in the fusion of receptor-containing vesicles with the cell surface. PMID:2409094

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and β-carotene significantly inhibited the antigen-induced release of β-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (FcϵRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited FcϵRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of FcϵRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of FcϵRI to lipid rafts, which are known as platforms of the aggregation of FcϵRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of FcϵRI to lipid rafts. This is the first report that focused on the aggregation of FcϵRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts. PMID:19700409

New precision medicine drugs oftentimes act through binding to specific cell-surface cancer receptors, and thus their efficacy is highly dependent on the availability of those receptors and the receptor concentration per cell. Paired-agent molecular imaging can provide quantitative information on receptor status in vivo, especially in tumor tissue; however, to date, published approaches to paired-agent quantitative imaging require that only ‘trace’ levels of imaging agent exist compared to receptor concentration. This strict requirement may limit applicability, particularly in drug binding studies, which seek to report on a biological effect in response to saturating receptors with a drug moiety. To extend the regime over which paired-agent imaging may be used, this work presents a generalized simplified reference tissue model (GSRTM) for paired-agent imaging developed to approximate receptor concentration in both non-receptor-saturated and receptor-saturated conditions. Extensive simulation studies show that tumor receptor concentration estimates recovered using the GSRTM are more accurate in receptor-saturation conditions than the standard simple reference tissue model (SRTM) (% error (mean ± sd): GSRTM 0 ± 1 and SRTM 50 ± 1) and match the SRTM accuracy in non-saturated conditions (% error (mean ± sd): GSRTM 5 ± 5 and SRTM 0 ± 5). To further test the approach, GSRTM-estimated receptor concentration was compared to SRTM-estimated values extracted from tumor xenograft in vivo mouse model data. The GSRTM estimates were observed to deviate from the SRTM in tumors with low receptor saturation (which are likely in a saturated regime). Finally, a general ‘rule-of-thumb’ algorithm is presented to estimate the expected level of receptor saturation that would be achieved in a given tissue provided dose and pharmacokinetic information about the drug or imaging agent being used, and physiological

The concept of an infectious agent playing a role in cardiovascular disease is slowly gaining attention. Among several pathogens identified, the oral bacterium Streptococcus gordonii has been implicated as a plausible agent. Platelet adhesion and subsequent aggregation are critical events in the pathogenesis and dissemination of the infective process. Here we describe the identification and characterization of a novel cell wall-anchored surface protein, PadA (397 kDa), of S. gordonii DL1 that binds to the platelet fibrinogen receptor GPIIbIIIa. Wild-type S. gordonii cells induced platelet aggregation and supported platelet adhesion in a GPIIbIIIa-dependent manner. Deletion of the padA gene had no effect on platelet aggregation by S. gordonii but significantly reduced (>75%) platelet adhesion to S. gordonii. Purified N-terminal PadA recombinant polypeptide adhered to platelets. The padA mutant was unaffected in production of other platelet-interactive surface proteins (Hsa, SspA, and SspB), and levels of adherence of the mutant to fetuin or platelet receptor GPIb were unaffected. Wild-type S. gordonii, but not the padA mutant, bound to Chinese hamster ovary cells stably transfected with GPIIbIIIa, and this interaction was ablated by addition of GPIIbIIIa inhibitor Abciximab. These results highlight the growing complexity of interactions between S. gordonii and platelets and demonstrate a new mechanism by which the bacterium could contribute to unwanted thrombosis. PMID:19884334

Tissue-engineering strategies may be employed in the development of in vitro breast tissue models for use in testing regimens of drug therapies and vaccines. The physical and chemical interactions that occur among cells and extracellular matrix components can also be elucidated with these models to gain an understanding of the progression of transformed epithelial cells into tumours and the ultimate metastases of tumour cells. The modified inkjet printer may be a useful tool for creating three-dimensional (3D) in vitro models, because it offers an inexpensive and high-throughput solution to microfabrication, and because the printer can be easily manipulated to produce varying tissue attributes. We hypothesized, however, that when ink is replaced with a biologically based fluid (i.e. a 'bio-ink'), specifically a serum-free cell culture medium, printer nozzle failure can result from salt scale build-up as fluid evaporates on the printhead surface. In this study, ethylene diamine tetra-acetic acid (EDTA) was used as a culture medium additive to prevent salt scaling and cell aggregation during the bioprinting process. The results showed that EDTA, at a concentration typically found in commercially available trypsin solutions (0.53 mM), prevented nozzle failure when a serum-free culture medium was printed from a nozzle at 1000 drops/s. Furthermore, increasing concentrations of EDTA appeared to mildly decrease aggregation of 4T07 cells. Cell viability studies were performed to demonstrate that addition of EDTA did not result in significant cell death. In conclusion, it is recommended that EDTA be incorporated into bio-ink solutions containing salts that could lead to nozzle failure.

One of the areas of active development of modern nanomedicine is drug/gene delivery and imaging application of nanoparticles functionalized by ligands, aptamers or antibodies capable of specific interactions with cell surfacereceptors. Being a complex multifunctional system different structural aspects of nanoparticles affect their interactions with cell surfaces and the surface properties of cells can be different (e.g. density, distribution and mobility of receptors). Computer simulations allow a systematic investigation of the influence of multiple factors and provide a unified platform for the comparison. Using Monte Carlo simulations we investigate the influence of the nanoparticle properties (nanoparticle size, polymer tether length, polydispersity, density, ligand energy, valence and density) on nanoparticle-cell surface interactions and make predictions regarding favorable nanoparticle design for achieving multiple ligand-receptor binding. We will also discuss the implications of nanoparticle design on the selectivity of attachment to cells with high receptor density while ``ignoring'' cells with a low density of receptors.

Cationic gemini surfactant homologues alkanediyl-α,ω-bis(dodecyldiethylammonium bromide), [C(12)H(25)(CH(3)CH(2))(2)N(CH(2))(S)N(CH(2)CH(3))(2)C(12)H(25)]Br(2) (where S=2, 4, 6, 8, 10, 12, 16, 20), referred to as C(12)C(S)C(12)(Et) were synthesized systematically. This paper focused on various properties of the above gemini surfactants in order to give a full understanding of this series of surfactants. The following points are covered: (1) surface properties, which include (i) effect of the spacer carbon number on the general properties and (ii) the effect of added NaBr on the general surface properties; (2) aggregation behavior in bulk solution, including (i) morphologies of above gemini surfactants classed as having short spacers, middle-length spacers and long spacers and (ii) superior vesicle stability against high NaBr concentration for the long spacer gemini surfactants; (3) thermodynamic properties during micellization and the effect of spacer carbon number on them; and (4) perspectives for the further use and application of these compounds.

Increases in expression of α4βδ GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3α-OH-5α[β]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether α4βδ trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces α4βδ expression in vivo. We first determined that flumazenil (100 nM – 100 μM, IC50=~1 μM) acted as a negative modulator, reducing GABA (10 μM)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant α4β2δ expressed in HEK-293 cells. Surface expression of recombinant α4β2δ receptors was detected using a 3XFLAG reporter at the C-terminus of α4 (α4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 μM) + THP (100 nM). Flumazenil (10 μM) decreased surface expression of α4F by ~60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of α4β2δ after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of α4β2δ were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of α4β2δ expression using flumazenil. PMID:26592470

Ligand binding to the nicotinic acetylcholine receptor is studied by surface plasmon resonance. Biotinylated bungarotoxin, immobilized on a streptavidin-coated gold film, binds nicotinic acetylcholine receptor both in detergent-solubilized and in lipid vesicle-reconstituted form with high specificity. In the latter case, nonspecific binding to the sensor surface is significantly reduced by reconstituting the receptor into poly(ethylene glycol)-lipid-containing sterically stabilized vesicles. By preincubation of a bulk nicotinic acetylcholine receptor sample with the competing ligands carbamoylcholine and decamethonium bromide, the subsequent specific binding of the receptor to the surface-immobilized bungarotoxin is reduced, depending on the concentration of competing ligand. This competition assay allows the determination of the dissociation constants of the acetylcholine receptor-carbamoylcholine complex. A K(D) = 3.5 × 10(-)(6) M for the detergent-solubilized receptor and a K(D) = 1.4 × 10(-)(5) M for the lipid vesicle-reconstituted receptor are obtained. For decamethonium bromide, a K(D) = 4.5 × 10(-)(5) M is determined for the detergent-solubilized receptor. This approach is of general importance for investigating ligand-receptor interactions in case of small ligand molecules by mass-sensitive techniques.

TARP [transmembrane AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor regulatory protein] γ-8 is an auxiliary subunit of AMPA receptors that is widely distributed in the hippocampus. It has been shown that TARP γ-8 promotes surface expression of AMPA receptors; however, how TARP γ-8 regulates the expression of AMPA receptors remains unclear. In the present study, we examined the effect of TARP glycosylation on AMPA receptor trafficking. We first showed that TARP γ-8 is an N-glycosylated protein, which contains two glycosylation sites, Asn53 and Asn56, and compared this with the glycosylation of TARP γ-2 and the AMPA receptor auxiliary protein CNIH-2 (cornichon homologue 2). We next examine the effect of TARP glycosylation on TARP trafficking and also on AMPA receptorsurface expression. We find that TARP γ-8 glycosylation is critical for surface expression of both TARP γ-8 and GluA1 in heterologous cells and neurons. Specifically, knockdown of TARP γ-8 causes a decrease in both total and surface AMPA receptors. We find that the expression of unglycosylated TARP γ-8 in cultured neurons is unable to restore GluA1 expression fully. Furthermore, when the maturation of TARP γ-8 is impaired, a large pool of immature GluA1 is retained intracellularly. Taken together, our data reveal an important role for the maturation of TARP γ-8 in the trafficking and function of the AMPA receptor complex.

It is hypothesized that desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) induced by chronic exposure to nicotine initiates upregulation of nAChR number. To test this hypothesis directly, oocytes expressing alpha4beta2 receptors were chronically incubated (24-48 hr) in nicotine, and the resulting changes in specific [3H]nicotine binding to surfacereceptors on intact oocytes were compared with functional receptor desensitization. Four lines of evidence strongly support the hypothesis. (1) The half-maximal nicotine concentration necessary to produce desensitization (9.7 nM) was the same as that needed to induce upregulation (9.9 nM). (2) The concentration of [3H]nicotine for half-maximal binding to surface nAChRs on intact oocytes was also similar (11.1 nM), as predicted from cyclical desensitization models. (3) Functional desensitization of alpha3beta4 receptors required 10-fold higher nicotine concentrations, and this was mirrored by a 10-fold shift in concentrations necessary for upregulation. (4) Mutant alpha4beta2 receptors that do not recover fully from desensitization, but not wild-type channels, were upregulated after acute (1 hr) applications of nicotine. Interestingly, the nicotine concentration required for half-maximal binding of alpha4beta2 receptors in total cell membrane homogenates was 20-fold lower than that measured for surface nAChRs in intact oocytes. These data suggest that cell homogenate binding assays may not accurately reflect the in vivo desensitization affinity of surface nAChRs and may account for some of the previously reported differences in the efficacy of nicotine for inducing nAChR desensitization and upregulation.

To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surfacereceptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surfacereceptors in living cells.

HiRISE images of Mars with ground sampling down to 25 cm/pixel show that the dust-rich mantle covering the surfaces of the Tharsis Montes is organized into ridges whose form and distribution are consistent with formation by aeolian saltation. Other dusty areas near the volcanoes and elsewhere on the planet exhibit a similar morphology. The material composing these "reticulate" bedforms is constrained by their remote sensing properties and the threshold curve combined with the saltation/suspension boundary, both of which vary as a function of elevation (atmospheric pressure), particle size, and particle composition. Considering all of these factors, dust aggregates are the most likely material composing these bedforms. We propose that airfall dust on and near the volcanoes aggregates in situ over time, maybe due to electrostatic charging followed by cementation by salts. The aggregates eventually reach a particle size at which saltation is possible. Aggregates on the flanks are transported downslope by katabatic winds and form linear and "accordion" morphologies. Materials within the calderas and other depressions remain trapped and are subjected to multidirectional winds, forming an interlinked "honeycomb" texture. In many places on and near the volcanoes, light-toned, low thermal inertia yardangs and indurated surfaces are present. These may represent "duststone" formed when aggregates reach a particle size below the threshold curve, such that they become stabilized and subsequently undergo cementation. ?? 2009 Elsevier Inc.

The impact of capping agents and environmental conditions (pH, ionic strength, and background electrolytes) on surface charge and aggregation potential of silver nanoparticles (AgNPs) suspensions were investigated. Capping agents are chemicals used in the synthesis of nanopartic...

Although many different receptors undergo endocytosis, the system-level design principles that govern the evolution of receptor dynamics are far from fully understood. We have constructed a generalized mathematical model to understand how receptor internalization dynamics encodes receptor function and regulation. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptors can be categorized a being: i) avidity-controlled where the response control depends primarily on the extracelluar ligand capture efficiency, ii) consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii) dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled and epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to anhance the accuracy of signaling receptors rather than serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulations.

The conjugative transfer of the Streptococcus faecalis plasmid pAD1 is characterized by a 10,000-fold increase in frequency following sex pheromone (cAD1) induction. Before the increase in plasmid transfer, donor cells synthesize a proteinaceous adhesin that facilitates the formation of mating aggregates. Four novel surface proteins appearing after exposure of pAD1-containing cells to sex pheromone have been identified. Thirty minutes after induction, a 130-kilodalton (kDa) protein was detectable by Western blotting. A 74-kDa protein, the major species present, and a pair of bands at 153 and 157 kDa were evident 45 min after induction. Induced cells containing another conjugative S. faecalis plasmid, pPD1, gave rise to three high-molecular-weight proteins of the same size (130, 153, and 157 kDa) as those synthesized by pAD1-containing cells. These proteins cross-reacted with antisera raised against induced cells containing pAD1. However, the major protein species produced by pPD1-containing cells had a molecular weight of 78,000 and did not cross-react significantly with the corresponding band of the pAD1 system. Pheromone-induced transfer of the two plasmids, when both were present in the same cell, was independent; induction was limited to the pheromone-specified plasmid. The possibility that lipoteichoic acid might act as a receptor (binding substance) for the induced adhesin protein was also explored. Free lipoteichoic acid (isolated from S. faecalis) inhibited clumping of induced cells, apparently by acting as a competitive inhibitor of the cellular binding substance. Images PMID:3093466

Retinal pigment epithelial cells, derived from human donor eyes, have been grown in culture as monolayers on membrane filters or plastic surfaces and shown to possess transferrin receptors with a monomeric molecular mass of 93,000. These receptors internalize 125I-labelled transferrin and recycle it to the surrounding medium in a similar manner to other cell types. Scatchard analyses show that there are about 100,000 high-affinity receptors on the surface of each cell and most of these receptors are associated with the cytoskeleton. In total cell extracts, there are additional low-affinity binding sites that do not appear to be strongly associated with the cytoskeleton. The apparent interaction of transferrin receptors with the cytoskeleton was confirmed in two ways: first, using 200 kV electron microscopy for stereo analyses, skeleton-associated transferrin receptors were detected by a monoclonal anti-receptor antibody and a colloidal gold-conjugated second antibody after Triton X-100 extraction of pigment epithelial cells grown directly on laminin-coated gold grids; and, second, when cell surfacereceptors were labelled with radioiodinated transferrin and then incubated for various periods of time, the labelled transferrin was observed to move from a Triton X-100-insoluble fraction (a putative cytoskeletal compartment) to a Triton-soluble compartment that was not associated with the cytoskeleton. Using either horseradish peroxidase or colloidal gold-labelled transferrin, it has been shown that basolateral and apical surface-located receptors participate in receptor-mediated endocytosis via clathrin-coated pits, endosomes and tubular structures. Initially, transferrin internalized from the apical surface is observed in small endosomes that often appear to be embedded in an apical layer of microfilaments. From these peripheral regions of the cells, the labelled receptors move to larger endosomes and multivesicular bodies deeper in the cytoplasm. These structures

Platelets are involved in host defense via clearance of bacteria from the circulation, interaction with virus particles, and uptake of various size particulates. There is a growing interest in micro- and nanoparticles for drug delivery and there is evidence that the properties of these particles critically influence their interaction and uptake by various tissues and cells including platelets. Virus mediated gene therapy applications are still challenged by the resultant thrombocytopenia and the mechanism(s) of platelet-foreign particles interaction remains unclear. We studied the specifics of platelet interaction with an active biological agent (adenovirus) and inert latex microspheres (MS) and investigated the role of platelet proteins in this interaction. We show that activated and not resting platelets internalize MS, without influencing platelet aggregation. In contrast, adenovirus induces and potentiates ADP-induced platelet aggregation and results in rapid expression of P-selectin. Platelets then internalize adenovirus and viral particles appear inside the open canalicular system. Inhibition of platelet αIIbβ3, GPIbα, and P-selectin decreases both platelet aggregation and internalization of MS. Inhibition of αIIbβ3 and αVβ3 does not abolish adenovirus platelet internalization and adenovirus-induced platelet activation is maintained. Our study demonstrates that platelets react differentially with foreign particles and that αIIbβ3 is a key player in platelet engulfing of foreign particles but not in mediating adenovirus internalization. Other platelet candidate molecules remain to be investigated as potential targets for management of adenovirus-induced thrombocytopenia.

We previously showed that dopamine receptors existed as homo- and heterooligomers, in cells and in brain tissue. We developed a method designed to study the formation and regulation of G protein coupled receptor (GPCR) oligomers in cells, using a GPCR into which a nuclear localization sequence (NLS) had been inserted. Unlike wildtype GPCRs, in the presence of agonist/antagonist ligands the GPCR-NLS is retained at the cell surface, and following ligand removal, the GPCR-NLS translocated from the cell surface. The D(1) dopamine receptor expressed with either D(2)-NLS or D(1-)NLS receptors translocated to the nucleus, indicating hetero- or homo-oligomerization with the NLS-containing receptor. Using these tools, we now demonstrate that D(1)-D(2) dopamine heterooligomers can be disrupted and the component receptors separated by dopamine and selective agonists that occupied one or both binding pockets. Subsequent agonist removal allowed the reformation of the heterooligomer. D(1) receptor homooligomers could also be disrupted by agonist, but at higher concentrations than that required for the disruption of the D(1)-D(2) heteromer. Dopamine D(1) or D(2) receptor antagonists had no effect on the integrity of the homo- or heterooligomer. We have also determined that the D(1)-D(2) heterooligomer contains D(1) homooligomers. These studies indicate that the populations of dopamine receptor oligomers at the cell surface are subject to conformational changes following agonist occupancy and are likely dynamically regulated following agonist activation.

The present investigation unravels the effect of trehalose on 1-hexadecyl-3-methylimidazolium chloride ([C16mim]Cl), a cationic surface active ionic liquid (SAIL) micelle and SAIL ([C16mim]Cl)-nonionic surfactant (Sorbitan Stearate, Span 60) based vesicles. The influence of trehalose on size and morphology of the aggregates has been investigated using dynamic light scattering (DLS) and transmission electron microscopic (TEM) measurements. Besides, we have studied the dynamic properties of curcumin inside these aggregates using fluorescence spectroscopic based techniques. The results revealed that trehalose molecules play crucial role in modulation of the photophysical properties of curcumin in these organized assemblies.

The environmental behaviors and risks associated with graphene have attracted considerable attention. However, the fundamental effects of ambient water and visible-light irradiation on the properties and toxicity of graphene remain unknown. This work revealed that hydration and irradiation result in the transformation of large-sheet graphene to long-ribbon graphene. The thickness of the treated graphene decreased, and oxides were formed through the generation of singlet oxygen. In addition, hydration and irradiation resulted in greater disorder in the graphene structure and in the expansion of the d-spacing of the structure due to the introduction of water molecules and modifications of the functional groups. Oxidative modifications with two-stage (fast and low) kinetics enhanced the number of negative surface charges on the graphene and enhanced graphene aggregation. The above property alterations reduced the nanotoxicity of graphene to algal cells by reducing the generation of reactive oxygen species, diminishing protein carbonylation and decreasing tail DNA. A comparative study using graphene oxide suggested that oxidative modifications could play an important role in inhibiting toxicological activity. This study provides a preliminary approach for understanding the environmental behaviors of graphene and avoids overestimating the risks of graphene in the natural environment.

A dynamic surface tension detector (DSTD) has been equipped with an additional pressure sensor for simultaneous viscosity measurements, as a detector for flow injection analysis. The viscosity measurement is based on a single capillary viscometer (SCV) placed in parallel configuration with the DSTD. The viscometer in the optimized conditions consists of a PEEK capillary (i.d.=0.25 mm, L=75 cm) kept at constant temperature using a thermostatic bath, which leads on the two sides to the two arms of a differential piezoelectric pressure transducer with a range of 0-35 psi. The DSTD, described previously, measures the changing pressure across the liquid/air interface of 2 μL drops repeatedly forming at the end of a capillary. SCV performance has been evaluated by measuring dynamic viscosity of water/glycerol mixtures analysed in flow injection and comparing the results with the values reported in the literature. The detection limits of SCV and DSTD, calculated as 3σ of the blank, were 0.012 cP and 0.6 dyn cm(-1), respectively. The FI-SCV-DSTD system has been applied to the study of temperature-induced denaturation/aggregation process in bovine serum albumin (BSA). The results have been supported and discussed with respect to BSA conformational analysis performed using Fourier Transform infrared spectroscopy.

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

The Shiga toxigenic Escherichia coli O104:H4 isolated during the 2011 European outbreak expresses Shiga toxin 2a and possess virulence genes associated with the enteroaggregative E. coli (EAEC) pathotype. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga-toxin adsorption, but it is not clear whether the AAF/I fimbriae are involved in the colonization and biofilm formation on food and environmental matrices such as the surface of fresh produce. We deleted the gene encoding for the AAF/I fimbriae main subunit (AggA) from an outbreak associated E. coli O104:H4 strain, and evaluated the role of AAF/I fimbriae in the adherence and colonization of E. coli O104:H4 to spinach and abiotic surfaces. The deletion of aggA did not affect the adherence of E. coli O104:H4 to these surfaces. However, it severely diminished the colonization and biofilm formation of E. coli O104:H4 on these surfaces. Strong aggregation and biofilm formation on spinach and abiotic surfaces were observed with the wild type strain but not the isogenic aggA deletion mutant, suggesting that AAF/I fimbriae play a crucial role in persistence of O104:H4 cells outside of the intestines of host species, such as on the surface of fresh produce.

Hyperglycemia and hyperlipidemia are considered critical to the development of diabetic nephropathy. The aim of this study is to clarify the effect of cholesterol on advanced-glycation-end-products and the mechanisms behind the advanced-glycation-end-product-cholesterol-aggregated bovine serum albumin (BSA)-induced proliferation of mesangial cells. Mesangial cells were treated with advanced-glycation-end-product-cholesterol-aggregated-BSA, and RNA and protein were isolated. Cholesterol caused a 1.5-fold increase in fluorescent intensity and 2-fold increase in advanced-glycation-end-products in vitro. Pyridoxamine, aminoguanidine, and N-acetyl-l-cycteine suppressed the production of advanced-glycation-end-product-cholesterol-aggregated-BSA. Advanced-glycation-end-product-cholesterol-BSA was analyzed by matrix-assisted-laser-desorption/ionization-time of flight mass spectrometry, and peaks were found to shift toward a higher mass. Advanced-glycation-end-product-cholesterol-aggregated-BSA induced overexpression of the mRNA of transforming growth factor-beta1, collagen type 1, collagen type 4 and receptor for advanced-glycation-end-products, and the proliferation of mesangial cells. The injection of advanced-glycation-end-product-cholesterol-aggregated-BSA caused glomerular changes and albuminuria in non-diabetic mice. A transforming-growth-factor-beta receptor 1 kinase inhibitor or Mitogen-activated-Protein-Kinase/Extracellular-Signal-regulated-Kinase kinase (ERK) inhibitor (U-0126) suppressed the proliferation of mesangial cells induced by advanced-glycation-end-product-cholesterol-aggregated-BSA dose-dependently. U-0126 inhibited the phosphorylation of ERK1/2 in advanced-glycation-end-product-cholesterol-aggregated-BSA treated mesangial cells. These findings suggested that cholesterol promotes the formation of advanced-glycation-end-products-protein and that advanced-glycation-end-product-cholesterol-aggregated protein stimulates mesangial cells to proliferate via

Streptococcus gordonii colonizes multiple sites within the human oral cavity. This colonization depends upon the initial interactions of streptococcal adhesins with host receptors. The adhesins that bind salivary agglutinin glycoprotein (gp340) and human cell surfacereceptors include the antigen I/II (AgI/II) family polypeptides SspA and SspB and a sialic acid-binding surface protein designated Hsa or GspB. In this study we determined the relative functions of the AgI/II polypeptides and Hsa in interactions of S. gordonii DL1 (Challis) with host receptors. For an isogenic mutant with the sspA and sspB genes deleted the levels of adhesion to surface-immobilized gp340 were reduced 40%, while deletion of the hsa gene alone resulted in >80% inhibition of bacterial cell adhesion to gp340. Adhesion of S. gordonii DL1 cells to gp340 was sialidase sensitive, verifying that Hsa has a major role in mediating sialic acid-specific adhesion to gp340. Conversely, aggregation of S. gordonii cells by fluid-phase gp340 was not affected by deletion of hsa but was eliminated by deletion of the sspA and sspB genes. Deletion of the AgI/II polypeptide genes had no measurable effect on hsa mRNA levels or Hsa surface protein expression, and deletion of hsa did not affect AgI/II polypeptide expression. Further analysis of mutant phenotypes showed that the Hsa and AgI/II proteins mediated adhesion of S. gordonii DL1 to human HEp-2 epithelial cells. Hsa was also a principal streptococcal cell surface component promoting adhesion of human platelets to immobilized streptococci, but Hsa and AgI/II polypeptides acted in concert in mediating streptococcal cell-platelet aggregation. The results suggest that Hsa directs primary adhesion events for S. gordonii DL1 (Challis) with immobilized gp340, epithelial cells, and platelets. AgI/II polypeptides direct gp340-mediated aggregation, facilitate multimodal interactions necessary for platelet aggregation, and modulate S. gordonii-host engagements

G protein coupled receptors (GPCRs) are one of the largest human gene families, and are targets for many important therapeutic drugs. Over the last few years, there has been a major paradigm shift in our understanding of how these receptors function. Formerly, GPCRs were thought to exist as monomers that, upon agonist occupation, activated a heterotrimeric G protein to alter the concentrations of specific second messengers. Until recently, this relatively linear cascade has been the standard paradigm for signaling by these molecules. However, it is now clear that this model is not adequate to explain many aspects of GPCR function. We now know that many, if not most, GPCRs form homo- and/or hetero-oligomeric complexes and interact directly with intracellular proteins in addition to G proteins. It now appears that many GPCRs may not function independently, but might more accurately be described as subunits of large multi-protein signaling complexes. These observations raise many important new questions; some of which include: 1) How many functionally and pharmacologically distinct receptor subtypes exist in vivo? 2) Which GPCRs physically associate, and in what stochiometries? 3) What are the roles of individual subunits in binding ligand and activating responses? 4) Are the pharmacological or signaling properties of GPCR heterodimers different from monomers? Since these receptors are the targets for a large number of clinically useful compounds, such information is likely to be of direct therapeutic importance, both in understanding how existing drugs work, but also in discovering novel compounds to treat disease. PMID:17011524

The issue of herb-drug interactions has been widely reported. Herbal ingredients can activate nuclear receptors and further induce the gene expression alteration of drug-metabolizing enzyme and/or transporter. Therefore, the herb-drug interaction will happen when the herbs and drugs are coadministered. This kind of interaction is called inductive herb-drug interactions. Pregnane X Receptor (PXR) and drug-metabolizing target genes are involved in most of inductive herb-drug interactions. To predict this kind of herb-drug interaction, the protocol could be simplified to only screen agonists of PXR from herbs because the relations of drugs with their metabolizing enzymes are well studied. Here, a combinational in silico strategy of pharmacophore modelling and docking-based rank aggregation (DRA) was employed to identify PXR's agonists. Firstly, 305 ingredients were screened out from 820 ingredients as candidate agonists of PXR with our pharmacophore model. Secondly, DRA was used to rerank the result of pharmacophore filtering. To validate our prediction, a curated herb-drug interaction database was built, which recorded 380 herb-drug interactions. Finally, among the top 10 herb ingredients from the ranking list, 6 ingredients were reported to involve in herb-drug interactions. The accuracy of our method is higher than other traditional methods. The strategy could be extended to studies on other inductive herb-drug interactions. PMID:26339628

In the macroactivity approach, dermal exposure is estimated using empirically-derived transfer coefficients to aggregate the mass transfer associated with a series of contacts with a contaminated medium. The macroactivity approach affords the possibility of developing screenin...

JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin’s enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin’s bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells. PMID:27300140

We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.

Fullerenes are carbon nanoparticles with widespread biomedical, commercial and industrial applications. Attributes such as their tendency to aggregate and aggregate size and shape impact their ability to be transported into and through the environment and living tissues. Knowledge of these properties is therefore valuable for their human and environmental risk assessment as well as to control their synthesis and manufacture. In this work, asymmetrical flow-field flow fractionation (AF4) coupled to multi-angle light scattering (MALS) was used for the first time to study the size distribution of surface modified fullerenes with both polyhydroxyl and carboxyl functional groups in aqueous solutions having different pH (6.5-11) and ionic strength values (0-200mM) of environmental relevance. Fractionation key parameters such as flow rates, flow programming, and membrane material were optimized for the selected fullerenes. The aggregation of the compounds studied appeared to be indifferent to changes in solution pH, but was affected by changes in the ionic strength. Polyhydroxy-fullerenes were found to be present mostly as 4nm aggregates in water without added salt, but showed more aggregation at high ionic strength, with an up to 10-fold increase in their mean hydrodynamic radii (200mM), due to a decrease in the electrostatic repulsion between the nanoparticles. Carboxy-fullerenes showed a much stronger aggregation degree in water (50-100nm). Their average size and recoveries decreased with the increase in the salt concentration. This behavior can be due to enhanced adsorption of the large particles to the membrane at high ionic strength, because of their higher hydrophobicity and much larger particle sizes compared to polyhydroxy-fullerenes. The method performance was evaluated by calculating the run-to-run precision of the retention time (hydrodynamic radii), and the obtained RSD values were lower than 1%. MALS measurements showed aggregate sizes that were in good

We have investigated the influence of Rabi splitting tuning on the dynamics of strongly coupled J-aggregate/surface plasmon polariton systems. In particular, the Rabi splitting was tuned by modifying the J-aggregate molecule concentration while a polaritonic system was provided by a nanostructure formed by holes array in a golden layer. From the periodic and concentration changes we have identified, through numerical and experimental steady-state analyses, the best geometrical configuration for maximizing Rabi splitting, which was then used for transient absorption measurements. It was found that in transient absorption spectra, under upper band excitation, two bleaching peaks appear when a nanostructured polaritonic pattern is used. Importantly, their reciprocal distance increases upon increase of J-aggregate concentration, a result confirmed by steady-state analysis. In a similar manner it was also found that the lifetime of the upper band is intimately related to the coupling strength. In particular, we argue that with strong coupling strength, i.e. high J-aggregate concentration, a short lifetime of the upper band has to be expected due to the suppression of the bottleneck effect. This result supports the idea that the dynamics of hybrid systems is profoundly dependent on Rabi splitting.

Aims: The aim of this work is to gain a deeper insight into how much different aggregate types are affected by erosion. Especially, it is important to study the influence of the velocity of the impacting projectiles. We also want to provide models for dust growth in protoplanetary disks with simple recipes to account for erosion effects. Methods: To study the erosion of dust aggregates we employed a molecular dynamics approach that features a detailed micro-physical model of the interaction of spherical grains. For the first time, the model has been extended by introducing a new visco-elastic damping force, which requires a proper calibration. Afterwards, different sample generation methods were used to cover a wide range of aggregate types. Results: The visco-elastic damping force introduced in this work turns out to be crucial to reproduce results obtained from laboratory experiments. After proper calibration, we find that erosion occurs for impact velocities of 5 ms-1 and above. Though fractal aggregates as formed during the first growth phase are most susceptible to erosion, we observe erosion of aggregates with rather compact surfaces as well. Conclusions: We find that bombarding a larger target aggregate with small projectiles results in erosion for impact velocities as low as a few ms-1. More compact aggregates suffer less from erosion. With increasing projectile size the transition from accretion to erosion is shifted to higher velocities. This allows larger bodies to grow through high velocity collisions with smaller aggregates.

The Toll-like receptor (TLR) family of pathogen recognition molecules has an important role in recognizing microbial pathogens and microbial breakdown products. Activation of TLRs in the corneal epithelium induces CXC chemokine production and recruitment of neutrophils to the corneal stroma. Although essential for pathogen killing, neutrophils can cause extensive tissue damage, leading to visual impairment and blindness. In this review, we examine the role of TLRs in microbial keratitis and in noninfectious corneal inflammation, most commonly associated with contact lens wear. We present recent findings on TLR signaling pathways in the cornea, including MyD88- and TRIF-dependent responses and discuss the role of resident macrophages and dendritic cells. Finally, we examine the potential for targeting the TLR pathway as a potential therapeutic intervention for microbial keratitis and contact lens-associated corneal inflammation. PMID:18781257

Cantilever arrays have been used to monitor biochemical interactions and their associated stress. However, it is often necessary to passivate the underside of the cantilever to prevent unwanted ligand adsorption, and this process requires tedious optimization. Here, we show a way to immobilize membrane receptors on nanomechanical cantilevers so that they can function without passivating the underlying surface. Using equilibrium theory, we quantitatively describe the mechanical responses of vancomycin, human immunodeficiency virus type 1 antigens and coagulation factor VIII captured on the cantilever in the presence of competing stresses from the top and bottom cantilever surfaces. We show that the area per receptor molecule on the cantilever surface influences ligand-receptor binding and plays an important role on stress. Our results offer a new way to sense biomolecules and will aid in the creation of ultrasensitive biosensors.

Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels. PMID:18261457

Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

In this study, we investigated whether persistent agonist stimulation of NTS2 receptors gives rise to down-regulation, in light of reports that their activation induced long-lasting effects. To address this issue, we incubated COS-7 cells expressing the rat NTS2 with neurotensin (NT) for up to 24 h and measured resultant cell surface [{sup 125}I]-NT binding. We found that NTS2-expressing cells retained the same surfacereceptor density despite efficient internalization mechanisms. This preservation was neither due to NTS2 neosynthesis nor recycling since it was not blocked by cycloheximide or monensin. However, it appeared to involve translocation of spare receptors from internal stores, as NT induced NTS2 migration from trans-Golgi network to endosome-like structures. This stimulation-induced regulation of cell surface NTS2 receptors was even more striking in rat spinal cord neurons. Taken together, these results suggest that sustained NTS2 activation promotes recruitment of intracellular receptors to the cell surface, thereby preventing functional desensitization.

Introduction Thrombosis is an invariable component contributing to cardiovascular events in patients with hypertension. One of the risk factors of cardiovascular disease is increased platelet activity. One among the widely used antihypertensive agents are Angiotensin II type 1 Receptor Blockers (ARBs). Even though there are many studies involving antihypertensive agents, their antithrombotic properties remain elusive and not fully characterized. Aim To evaluate the anti-aggregatory effect of ARBs on platelets in-vivo. Materials and Methods A total of 60 subjects were included in this observational pilot study conducted in the medicine out patient department of JJM Hospital, Davanagere, Karnataka, India. Among them, 30 patients with essential hypertension attending Medicine OPD of a tertiary care hospital, who were on ARB for at least one month, were enrolled into study group. The control group consisted of 30 normotensive subjects who were not on any drug affecting platelet function. The Bleeding Time (BT) was evaluated for both the groups using Duke method of BT estimation. Data was analysed using SPSS software version 20. The test group was compared with control group using student’s unpaired t-test. Results The mean BT of study group was 2.488 minutes ± 0.0361 Standard Error of Mean (SEM) and that of control group was 1.998 minutes ± 0.0362 SEM. The result was statistically significant (p<0.001). The average duration of treatment was 2.933 years. Conclusion ARB have antiplatelet activity. Increase in BT in ARB group when compared with that of control group is a reflection of antiplatelet activity. PMID:28050394

Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism.

Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB2 and immobilization at solid interfaces. Expression of CB2 as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB2 was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB2 from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB2 and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB2.

The amino acid sequences of nicotinic acetylcholine receptors (nAChRs) from diverse species can be compared across extracellular, transmembrane, and intracellular domains. The intracellular domains are most divergent among subtypes, yet relatively consistent among species. The diversity indicates that each nAChR subtype possesses a unique language for communication with its host cell. The conservation across species also suggests that the intracellular domains may play defining functional roles for each subtype. Secondary structure prediction indicates two relatively conserved alpha helices within the intracellular domains of all nAChRs. Among all subtypes, the intracellular domain of α7 nAChR is one of the most-well conserved, and α7 nAChRs have effects in non-neuronal cells independent of generating ion currents, making it likely that the α7 intracellular domain directly mediates signal transduction. There are potential phosphorylation and protein binding sites in the α7 intracellular domain, which are conserved and may be the basis for α7-mediated signal transduction. PMID:26067101

CoCr alloy is commonly used in various cardiovascular medical devices for its excellent physical and mechanical properties. However, the formation of blood clots on the alloy surfaces is a serious concern. This research is focused on the surface modification of CoCr alloy using varying concentrations (1, 25, 50, 75, and 100 mM) of phosphoric acid (PA) and phosphonoacetic acid (PAA) to generate various surfaces with different wettability, chemistry, and roughness. Then, the adsorption of blood plasma proteins such as albumin and fibrinogen and the adhesion, activation, and aggregation of platelets with the various surfaces generated were investigated. Contact angle analysis showed PA and PAA coatings on CoCr provided a gradient of hydrophilic surfaces. FTIR showed PA and PAA were covalently bound to CoCr surface and formed different bonding configurations depending on the concentrations of coating solutions used. AFM showed the formation of homogeneous PA and PAA coatings on CoCr. The single and dual protein adsorption studies showed that the amount of albumin and fibrinogen adsorbed on the alloy surfaces strongly depend on the type of PA and PAA coatings prepared by different concentrations of coating solutions. All PA coated CoCr showed reduced platelet adhesion and activation when compared to control CoCr. Also, 75 and 100 mM PA-CoCr showed reduced platelet aggregation. For PAA coated CoCr, no significant difference in platelet adhesion and activation was observed between PAA coated CoCr and control CoCr. Thus, this study demonstrated that CoCr can be surface modified using PA for potentially reducing the formation of blood clots and improving the blood compatibility of the alloy.

ABSTRACT Enterovirus 71 (EV71) is a highly transmissible pathogenic agent that causes severe central nervous system diseases in infected infants and young children. Here, we reported that EV71 VP1 protein could bind to vimentin intermediate filaments expressed on the host cell surface. Soluble vimentin or an antibody against vimentin could inhibit the binding of EV71 to host cells. Accompanied with the reduction of vimentin expression on the cell surface, the binding of EV71 to cells was remarkably decreased. Further evidence showed that the N terminus of vimentin is responsible for the interaction between EV71 and vimentin. These results indicated that vimentin on the host cell surface may serve as an attachment site that mediated the initial binding and subsequently increased the infectivity of EV71. IMPORTANCE This study delivers important findings on the roles of vimentin filaments in relation to EV71 infection and provides information that not only improves our understanding of EV71 pathogenesis but also presents us with potentially new strategies for the treatment of diseases caused by EV71 infections. PMID:24623428

Monoclonal-nonspecific suppressor factor (MNSF) is a lymphokine derived from murine T cell hybridoma. The target tissues are both LPS-stimulated B cells and Con A-stimulated T cells. Since the action of MNSF may be mediated by its binding to specific cell surfacereceptors, we characterized the mode of this binding. The purified MNSF was labeled with {sup 125}I, using the Bolton-Hunter reagent. The labeled MNSF bound specifically to a single class of receptor (300 receptors per cell) on mitogen-stimulated murine B cells or T cells with an affinity of 16 pM at 24{degrees}C, in the presence of sodium azide. Competitive experiments showed that MNSF bound to the specific receptor and that the binding was not shared with IL2, IFN-gamma, and TNF. Various cell types were surveyed for the capacity to specifically bind {sup 125}I-MNSF. {sup 125}I-MNSF bound to MOPC-31C (a murine plasmacytoma line) and to EL4 (a murine T lymphoma line). The presence of specific binding correlates with the capacity of the cells to respond to MNSF. These data support the view that like other polypeptide hormones, the action of MNSF is mediated by specific cell surface membrane receptor protein. Identification of these receptors will provide insight into the apparently diverse activities of MNSF.

Transmembrane proteins are continuously shuttled from the endosomal compartment to the neuronal plasma membrane by highly regulated and complex trafficking steps. These events are involved in many homeostatic and physiological processes such as neuronal growth, signaling, learning and memory among others. We have previously shown that endosomal exocytosis of the B2 adrenergic receptor (B2AR) and the GluR1-containing AMPA receptor to the neuronal plasma membrane is mediated by two different types of vesicular fusion. A rapid type of exocytosis in which receptors are delivered to the plasma membrane in a single kinetic step, and a persistent mode in which receptors remain clustered at the insertion site for a variable period of time before delivery to the cell surface. Here, by comparing the exocytosis of multiple receptors in dissociated hippocampal and striatal cultures, we show that persistent events are a general mechanism of vesicular delivery. Persistent events were only observed after 10 days in vitro, and their frequency increased with use of the calcium ionophore A23187 and with depolarization induced by KCl. Finally, we determined that vesicles producing persistent events remain at the plasma membrane, closing and reopening their fusion pore for a consecutive release of cargo in a mechanism reminiscent of synaptic kiss-and-run. These results indicate that the delivery of transmembrane receptors to the cell surface can be dynamically regulated by kiss-and-run exocytosis. PMID:25404895

Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.

We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures. PMID:8926122

The aim of the present study was to analyze the surface expression of the NMDA-like receptors during the consolidation of contextual learning in the crab Neohelice granulata Memory storage is based on alterations in the strength of synaptic connections between neurons. The glutamatergic synapses undergo various forms of N-methyl-D aspartate receptor (NMDAR)-dependent changes in strength, a process that affects the abundance of other receptors at the synapse and underlies some forms of learning and memory. Here we propose a direct regulation of the NMDAR. Changes in NMDAR's functionality might be induced by the modification of the subunit's expression or cellular trafficking. This trafficking does not only include NMDAR's movement between synaptic and extra-synaptic localizations but also the cycling between intracellular compartments and the plasma membrane, a process called surface expression. Consolidation of contextual learning affects the surface expression of the receptor without affecting its general expression. The surface expression of the GluN1 subunit of the NMDAR is down-regulated immediately after training, up-regulated 3 h after training and returns to naïve and control levels 24 h after training. The changes in NMDAR surface expression observed in the central brain are not seen in the thoracic ganglion. A similar increment in surface expression of GluN1 in the central brain is observed 3 h after administration of the competitive GABAA receptor antagonist, bicuculline. These consolidation changes are part of a plasticity event that first, during the down-regulation, stabilizes the trace and later, at 3-h post-training, changes the threshold for synapse activation.

MCF-7 human breast cancer cells express E-cadherin and show, at least in some circumstances, E-cadherin-dependent cell-cell adhesion (Bracke et al., 1993). The MCF-7/AZ variant spontaneously displays E-cadherin-dependent fast aggregation; in the MCF-7/6 variant, E-cadherin appeared not to be spontaneously functional in the conditions of the fast aggregation assay, but function could be induced by incubation of the suspended cells in the presence of insulinlike growth factor I (IGF-I) (Bracke et al., 1993). E-cadherin from MCF-7 cells was shown to contain sialic acid. Treatment with neuraminidase was shown to remove this sialic acid, as well as most of the sialic acid present at the cell surface. Applied to MCF-7/AZ, and MCF-7/6 cells, pretreatment with neuraminidase abolished spontaneous as well as IGF-I induced, E-cadherin-dependent fast cell-cell adhesion of cells in suspension, as measured in the fast aggregation assay. Treatment with neuraminidase did not, however, inhibit the possibly different, but equally E-cadherin-mediated, process of cell-cell adhesion of MCF-7 cells on a flat plastic substrate as assessed by determining the percentage of cells remaining isolated (without contact with other cells) 24 h after plating.

The aim of the present study was to analyze the surface expression of the NMDA-like receptors during the consolidation of contextual learning in the crab "Neohelice granulata". Memory storage is based on alterations in the strength of synaptic connections between neurons. The glutamatergic synapses undergo various forms of…

Zeolite analcime with a core-shell and hollow icositetrahedron architecture was prepared by a one-pot hydrothermal route in the presence of ethylamine and Raney Ni. Detailed investigations on samples at different preparation stages revealed that the growth of the complex single crystalline geometrical structure did not follow the classic crystal growth route, i.e., a crystal with a highly symmetric morphology (such as polyhedra) is normally developed by attachment of atoms or ions to a nucleus. A reversed crystal growth process through oriented aggregation of nanocrystallites and surface recrystallization was observed. The whole process can be described by the following four successive steps. (1) Primary analcime nanoplatelets undergo oriented aggregation to yield discus-shaped particles. (2) These disci further assemble into polycrystalline microspheres. (3) The relatively large platelets grow into nanorods by consuming the smaller ones, and meanwhile, the surface of the microspheres recrystallizes into a thin single crystalline icositetrahedral shell via Ostwald ripening. (4) Recrystallization continues from the surface to the core at the expense of the nanorods, and the thickness of the monocrystalline shell keeps on increasing until all the nanorods are consumed, leading to hollow single crystalline analcime icositetrahedra. The present work adds new useful information for the understanding of the principles of zeolite growth.

The extracellular matrix (ECM) is a source of bioactive fragments called matricryptins or matrikines resulting from the proteolytic cleavage of extracellular proteins (e.g., collagens, elastin, and laminins) and proteoglycans (e.g., perlecan). Matrix metalloproteinases (MMPs), cathepsins, and bone-morphogenetic protein-1 release fragments, which regulate physiopathological processes including tumor growth, metastasis, and angiogenesis, a pre-requisite for tumor growth. A number of matricryptins, and/or synthetic peptides derived from them, are currently investigated as potential anti-cancer drugs both in vitro and in animal models. Modifications aiming at improving their efficiency and their delivery to their target cells are studied. However, their use as drugs is not straightforward. The biological activities of these fragments are mediated by several receptor families. Several matricryptins may bind to the same matricellular receptor, and a single matricryptin may bind to two different receptors belonging or not to the same family such as integrins and growth factor receptors. Furthermore, some matricryptins interact with each other, integrins and growth factor receptors crosstalk and a signaling pathway may be regulated by several matricryptins. This forms an intricate 3D interaction network at the surface of tumor and endothelial cells, which is tightly associated with other cell-surface associated molecules such as heparan sulfate, caveolin, and nucleolin. Deciphering the molecular mechanisms underlying the behavior of this network is required in order to optimize the development of matricryptins as anti-cancer agents. PMID:26869928

ABSTRACT Virus binding to the cell surface triggers an array of host responses, including activation of specific signaling pathways that facilitate steps in virus entry. Using mouse polyomavirus (MuPyV), we identified host signaling pathways activated upon virus binding to mouse embryonic fibroblasts (MEFs). Pathways activated by MuPyV included the phosphatidylinositol 3-kinase (PI3K), FAK/SRC, and mitogen-activated protein kinase (MAPK) pathways. Gangliosides and α4-integrin are required receptors for MuPyV infection. MuPyV binding to both gangliosides and the α4-integrin receptors was required for activation of the PI3K pathway; however, either receptor interaction alone was sufficient for activation of the MAPK pathway. Using small-molecule inhibitors, we confirmed that the PI3K and FAK/SRC pathways were required for MuPyV infection, while the MAPK pathway was dispensable. Mechanistically, the PI3K pathway was required for MuPyV endocytosis, while the FAK/SRC pathway enabled trafficking of MuPyV along microtubules. Thus, MuPyV interactions with specific cell surfacereceptors facilitate activation of signaling pathways required for virus entry and trafficking. Understanding how different viruses manipulate cell signaling pathways through interactions with host receptors could lead to the identification of new therapeutic targets for viral infection. PMID:27803182

Helicobacter pylori (H. pylori), a major cause of gastroduodenal diseases, produces VacA, a vacuolating cytotoxin associated with gastric inflammation and ulceration. The C-terminal domain of VacA plays a crucial role in receptor recognition on target cells. We have previously identified three proteins (i.e., RPTPα, RPTPβ, and LRP1) that serve as VacA receptors. These receptors contribute to the internalization of VacA into epithelial cells, activate signal transduction pathways, and contribute to cell death and gastric ulceration. In addition, other factors (e.g., CD18, sphingomyelin) have also been identified as cell-surface, VacA-binding proteins. Since we believe that, following interactions with its host cell receptors, VacA participates in events leading to disease, a better understanding of the cellular function of VacA receptors may provide valuable information regarding the mechanisms underlying the pleiotropic actions of VacA and the pathogenesis of H. pylori-mediated disease. In this review, we focus on VacA receptors and their role in events leading to cell damage. PMID:27187473

Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor. PMID:23097443

Fluorescence cell imaging can be used for disease diagnosis and cellular signal transduction. Using a metal nanoshell as molecular imaging agent, we develop a cellular model system to detect CXCR4 chemokine receptor on T-lymphatic cell surface. These metal nanoshells are observed to express enhanced emission intensity and shortened lifetimes due to the near-field interactions. They are covalently bound with anti-CXCR4 monoclonal antibodies for immunoreactions with the target sites of the CXCR4 receptors on the CEM-SS cells. The fluorescence intensity and lifetime cell images are recorded with a time-resolved confocal microscopy. As expected, the emission signals from the metal nanoshells are clearly isolated from the cellular autofluorescence due to strong intensities and distinctive lifetimes. The number of emission spots on the single cell image is estimated by direct count to the emission signals. Analyzing a pool of cell images, a maximal count number is obtained in a range of 200+/-50. Because there is an average of ~6000 binding sites on the cell surface, we estimate that one emission spot from the metal nanoshell may represent ~30 CXCR5 receptors. In addition, the CXCR4 receptors are estimated to distribute on ~70% area of the cell surface.

It has been shown that the establishment of urinary tract infection by Escherichia coli is dependent on attachment of the bacteria to epithelial cells1-4. The attachment involves specific epithelial cell receptors, which have been characterized as glycolipids5-10. Reversible binding to cell-surface mannosides may also be important4,11-13. This suggests an approach to the treatment of infections-that of blocking bacterial attachment with cell membrane receptor analogues. Using E. coli mutants lacking one or other of the two binding specificities (glycolipid and mannose), we show here that glycolipid analogues can block in vitro adhesion and in vivo urinary tract infection.

Upon activation, the epidermal growth factor (EGF) receptor becomes phosphorylated and triggers a vast signaling network that has profound effects on cell growth. The EGF receptor is observed to assemble into clusters after ligand binding and tyrosine kinase autophosphorylation, but the role of these assemblies in the receptor signaling pathway remains unclear. To address this question, we measured the phosphorylation of EGFR when the EGF ligand was anchored onto laterally mobile and immobile surfaces. We found that cells generated clusters of ligand-receptor complex on mobile EGF surfaces, and generated a lower ratio of phosphorylated EGFR to EGF than when compared to immobilized EGF that is unable to cluster. This result was verified by tuning the lateral assembly of ligand-receptor complexes on the surface of living cells using patterned supported lipid bilayers. Nanoscale metal lines fabricated into the supported membrane constrained lipid diffusion and EGF receptor assembly into micron and sub-micron scale corrals. Single cell analysis indicated that clustering impacts EGF receptor activation, and larger clusters (> 1 m2) of ligand-receptor complex generated lower EGF receptor phosphorylation per ligand than smaller assemblies (< 1 m2) in HCC1143 cells that were engaged to ligand-functionalized surfaces. We investigated EGFR clustering by treating cells with compounds that disrupt the cytoskeleton (Latrunculin-B), clathrin-mediated endocytosis (Pitstop2), and inhibit EGFR activation (Gefitinib). These results help elucidate the nature of large-scale EGFR clustering, thus underscoring the general significance of receptor spatial organization in tuning function.

Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency. PMID:25637053

One of the major challenges in the complete resection of cancer is the difficulty of distinctly classifying tumor and healthy tissue. This paper investigates the capability of competing kinetic modeling approaches for identifying different tissue types based on differential cell-surfacereceptor expressions. These approaches require fresh resected tissues to be stained with a mixture of two probes: one targeted to a cancer specific cell-surfacereceptor, and another left "untargeted" to account for nonspecific retention of the targeted agent, with subsequent repeated rinsing and imaging of the probe concentrations. Analysis of the results were carried out in simulations and in animal experiments for the cancer target, epidermal growth factor receptor (EGFR), a cell surfacereceptor overexpressed by many cancers. In the animal experiments, subcutaneous xenografts of human glioma (U251; moderate EGFR) and human epidermoid (A431; high EGFR) tumors, grown in six athymic mice, were excised and stained with an EGFR targeted surface-enhanced Raman scattering nanoparticle (SERS NP) and untargeted SERS NP pair. The salient finding in this study was that significant non-specific retention was observed for the EGFR targeted probe [anti-EGFR antibody labeled with a surface-enhanced Raman scattering (SERS) nanoparticle], but could be corrected for by the equivalent non-specific retention of the untargeted probe (isotype control antibody labeled with a different SERS nanoparticle). Once this non-specific binding was accounted for, the kinetic model was able to predict the expected differences in EGFR concentration among different tissue types: healthy, U251, and A431 in accordance with an ex vivo flow cytometry analysis, successfully classifying different tissue types.

Aim: Studies of the α7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of α7-type AChRs1. The current work aims at obtaining and characterizing a cell line with high functional expression of the human α7 AChR. Methods: Ric-3 cDNA was incorporated into SHE-P1-hα7 cells expressing the α7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [125I]α-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent α-bungarotoxin, anti-α7 antibody, and GFP-α7 were performed on the new clone. Results: The human α7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-hα7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [125I]αBTX saturable binding with an apparent KD of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-P1-hα7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-α7 antibodies. In contrast, chaperone-less SHE-P1-hα7 cells expressed only intracellular α7 AChRs and failed to produce detectable single-channel currents. Conclusion: The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal α7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells. PMID:19498422

In order to minimize the forward contamination of Mars, spacecraft are assembled under clean-room conditions that often require several procedures to clean and sterilize components. Surface characteristics of spacecraft materials may contribute to microbial survival by protecting spores from sterilizing agents, including UV irradiation on the surface of Mars. The primary objective of this study was to evaluate the effects of surface characteristics of several spacecraft materials on the survival of Bacillus subtilis spores under simulated Martian conditions.

The seco-prezizaane-type sesquiterpenes pseudoanisatin and parviflorolide from Illicium are noncompetitive antagonists at housefly (Musca domestica) gamma-aminobutyric acid (GABA) receptors. They show selectivity toward the insect receptor and thus represent new leads toward selective insecticides. Based on the binding data for 13 seco-prezizaane terpenoids and 17 picrotoxane and picrodendrane-type terpenoids to housefly and rat GABA receptors, a QSAR study was conducted by quasi-atomistic receptor-surface modeling (Quasar). The resulting models provide insight into the structural basis of selectivity and properties of the binding sites at GABA receptor-coupled chloride channels of insects and mammals.

Humic substances (HS) form molecular aggregates in solution and on mineral surfaces. Elucidation of the mechanism of formation of these aggregates is important for an understanding of the interactions of HS in soils arid natural waters. The HS are formed mainly by enzymatic depolymerization and oxidation of plant biopolymers. These reactions transform the aromatic and lipid plant components into amphiphilic molecules, that is, molecules that consist of separate hydrophobic (nonpolar) and hydrophilic (polar) parts. The nonpolar parts of the molecules are composed of relatively unaltered segments of plant polymers and the polar parts of carboxylic acid groups. These amphiphiles form membrane-like aggregates on mineral surfaces and micelle-like aggregates in solution. The exterior surfaces of these aggregates are hydrophilic, and the interiors constitute separate hydrophobic liquid-like phases.

The steroidogenic acute regulatory protein (StAR) is responsible for acute control of cholesterol transport across the mitochondrial membrane, however the mechanism of StAR-associated cholesterol transport is unknown and may involve the peripheral-type benzodiazepine receptor (PBR)/endozepine system. Several molecules of PBR may associate to form a channel through which cholesterol passes to the inner mitochondrial membrane, and endozepine is the natural ligand for PBR. Bioluminescence resonance energy transfer (BRET) was used to test StAR/PBR/endozepine interactions, PBR aggregation, and the effect of second messengers on interactions. There was no evidence of StAR/PBR, StAR/endozepine, or PBR/endozepine interactions. The StAR and PBR fusion proteins were trafficking to the mitochondria as expected, but the endozepine fusion protein was not localized to the mitochondria indicating that it was not biologically active. Data were obtained indicating that PBR forms aggregates in the mitochondrial membrane. Energy transfer between PBR fusion proteins was dose and time dependent, but there was no effect induced by PK11195 ligand binding or pharmacologic activation of PKA or PKC second messenger pathways. It appears that PBR aggregates in the mitochondrial membrane, however there was no evidence that PBR aggregation is regulated in the acute control of steroidogenesis, or that PBR and StAR interact.

Autoantibodies directed against the cell surfacereceptors for insulin are found in some patients with extreme insulin resistance. These antibodies specifically inhibit the binding of insulin to its receptor. A purified IgG fraction from one patient's plasma was labeled with 125I. The 125I-labeled antireceptor antibody, which initially represented about 0.3% of the total 125I-IgG, was enriched by selective adsorption and subsequent elution from cells rich in insulin receptors. The 125I-antireceptor antibody bound to cells and the binding was inhibited by whole plasma and purified IgG from this patient, as well as whole plasma from another patient with autoantibodies to the insulin receptor. Insulins that differed 300-fold in biological potency and affinity inhibited binding of 125I-antireceptor antibody in direct proportion to their ability to bind to the insulin receptor. The binding of 125I-antireceptor antibody was closely correlated with the binding of 125I-insulin over a wide range of receptor concentrations on different cell types. Experimentally induced reduction of the insulin receptor concentration was associated with parallel decreases in the binding of 125I-antireceptor antibody and 125I-insulin. The preparation of 125I-antireceptor antibody with a high specific activity by cytoadsorption and elution has provided a sensitive method for the detection of receptors and autoantibodies to cell surface components. PMID:1069300

Macrophage migration inhibitory factor (MIF) is recognized as a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. Our group has identified both chicken and Eimeria MIFs, and characterized their function in enhancing innate immune responses during inflammation. In this study, we report that chicken CD74 (ChCD74), a type II transmembrane protein, functions as a macrophage surfacereceptor that binds to MIF molecules. First, to examine the binding of MIF to chicken monocytes/macrophages, fresh isolated chicken peripheral blood mononuclear cells (PBMCs) were stimulated with rChIFN-γ and then incubated with recombinant chicken MIF (rChMIF). Immunofluorescence staining with anti-ChMIF followed by flow cytometry revealed the binding of MIF to stimulated PBMCs. To verify that ChCD74 acts as a surfacereceptor for MIF molecules, full-length ChCD74p41 was cloned, expressed and its recombinant protein (rChCD74p41) transiently over-expressed with green fluorescent protein in chicken fibroblast DF-1 cells. Fluorescence analysis revealed a higher population of cells double positive for CD74p41 and rChMIF, indicating the binding of rChMIF to DF-1 cells via rChCD74p41. Using a similar approach, it was found that Eimeria MIF (EMIF), which is secreted by Eimeria sp. during infection, bound to chicken macrophages via ChCD74p41 as a surfacereceptor. Together, this study provides conclusive evidence that both host and parasite MIF molecules bind to chicken macrophages via the surfacereceptor ChCD74.

Cannabinoid receptor type 2 (CB2) is the primary receptor pathway mediating the immunologic consequences of cannabinoids. We recently reported that human peripheral blood B cells express CB2 on both the extracellular membrane and at intracellular sites, where-as monocytes and T cells only express intracellular CB2. To better understand the pattern of CB2 expression by human B cells, we examined CD20(+) B cells from three tissue sources. Both surface and intracellular expression were present and uniform in cord blood B cells, where all cells exhibited a naïve mature phenotype (IgD(+)/CD38(Dim)). While naïve mature and quiescent memory B cells (IgD(-)/CD38(-)) from tonsils and peripheral blood exhibited a similar pattern, tonsillar activated B cells (IgD(-)/CD38(+)) expressed little to no surface CB2. We hypothesized that regulation of the surface CB2 receptor may occur during B cell activation. Consistent with this, a B cell lymphoma cell line known to exhibit an activated phenotype (SUDHL-4) was found to lack cell surface CB2 but express intracellular CB2. Furthermore, in vitro activation of human cord blood resulted in a down-regulation of surface CB2 on those B cells acquiring the activated phenotype but not on those retaining IgD expression. Using a CB2 expressing cell line (293 T/CB2-GFP), confocal microscopy confirmed the presence of both cell surface expression and multifocal intracellular expression, the latter of which co-localized with endoplasmic reticulum but not with mitochondria, lysosomes, or nucleus. Our findings suggest a dynamic multi-compartment expression pattern for CB2 in B cells that is specifically modulated during the course of B cell activation.

Surface charges on nanoscale structures in liquids, such as biomolecules and nano-micelles, play an essentially important role in their structural stability as well as their chemical activities. These structures interact with each other through electric double layers (EDLs) formed by the counter ions in electrolyte solution. Although static-mode atomic force microscopy (AFM) including colloidal-probe AFM is a powerful technique for surface charge density measurements and EDL analysis on a submicron scale in liquids, precise surface charge density analysis with single-nanometer resolution has not been made because of its limitation of the resolution and the detection sensitivity. Here we demonstrate molecular-scale surface charge measurements of self-assembled micellar structures, molecular hemicylinders of sodium dodecyl sulfate (SDS), by three-dimensional (3D) force mapping based on frequency modulation AFM. The SDS hemicylindrical structures with a diameter of 4.8 nm on a graphite surface were clearly imaged. We have succeeded in visualizing 3D EDL forces on the SDS hemicylinder surfaces and obtaining the molecular-scale charge density for the first time. The results showed that the surface charge on the trench regions between the hemicylinders was much smaller than that on the hemicylinder tops. The method can be applied to a wide variety of local charge distribution studies, such as spatial charge variation on a single protein molecule.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

Introduction: The aim of this study was to compare the shear bond strength of resin modified glass ionomer cement to conditioned and unconditioned mineral trioxide aggregatesurface. Materials and Method: White Mineral Trioxide Aggregate (WMTA) and Resin Modified Glass Ionomer Cement (RMGIC) were used for the study. 60 WMTA specimens were prepared and stored in an incubator at 37° C and 100% humidity for 72 hrs. The specimens were then divided into two groups- half of the specimens were conditioned and remaining half were left unconditioned, subsequent to which RMGIC was placed over MTA. The specimens were then stored in an incubator for 24 hrs at 37° C and 100% humidity. The shear bond strength value of RMGIC to conditioned and unconditioned WMTA was measured and compared using unpaired 't ’ test. Results: The mean shear bond strength of value of RMGIC to conditioned and unconditioned WMTA was 6.59 MPa and 7.587 MPa respectively. Statistical analysis using unpaired t-test revealed that the difference between values of two groups was not statistically significant (P > 0.05). Conclusions: During clinical procedures like pulp capping and furcal repair, if RMGIC is placed as a base over MTA, then conditioning should be done to increase the bond strength between RMGIC and dentin and any inadvertent contact of conditioner with MTA will not significantly affect the shear bond strength value of RMGIC to MTA. PMID:25298644

A novel concept is proposed for converting liquid-phase colorimetric assay into enhanced surface-tethered electrochemical analysis, which is based on the analyte-induced formation of a network architecture of metal nanoparticles (MNs). In a proof-of-concept trial, thymine-functionalized silver nanoparticle (Ag-T) is designed as the sensing unit for Hg(2+) determination. Through a specific T-Hg(2+)-T coordination, the validation system based on functionalized sensing units not only can perform well in a colorimetric Hg(2+) assay, but also can be developed into a more sensitive and stable electrochemical Hg(2+) sensor. In electrochemical analysis, the simple principle of analyte-induced aggregation of MNs can be used as a dual signal amplification strategy for significantly improving the detection sensitivity. More importantly, those numerous and diverse colorimetric assays that rely on the target-induced aggregation of MNs can be augmented to satisfy the ambitious demands of sensitive analysis by converting them into electrochemical assays via this approach.

The primary objective of this study was to evaluate the effects of surface characteristics of several spacecraft materials on the survival of Bacillus subtilis spores under simulated martian conditions.

Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression. PMID:26963253

Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a main marker of the presence of degenerative disorders such as amyloidoses. To elucidate the mechanisms of protein misfolding, the interaction of proteins with inorganic surfaces is of particular relevance, since surfaces displaying different wettability properties may represent model systems of the cell membrane. Here, we unveil the role of surface hydrophobicity/hydrophilicity in the misfolding of the Josephin domain (JD), a globular-shaped domain of ataxin-3, the protein responsible for the spinocerebellar ataxia type 3. By means of a combined experimental and theoretical approach based on atomic force microscopy, Fourier transform infrared spectroscopy and molecular dynamics simulations, we reveal changes in JD morphology and secondary structure elicited by the interaction with the hydrophobic gold substrate, but not by the hydrophilic mica. Our results demonstrate that the interaction with the gold surface triggers misfolding of the JD when it is in native-like configuration, while no structural modification is observed after the protein has undergone oligomerization. This raises the possibility that biological membranes would be unable to affect amyloid oligomeric structures and toxicity. PMID:23527026

Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) is a membrane bound protein that has been associated with a variety of solid tumors and the control of cell survival, proliferation, and metabolism. Quantification of the EGFR expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis and treatment. Here we report mapping of the distribution and interaction kinetics of EGFR in their native environment with the surface plasmon resonance imaging (SPRi) technique. The monoclonal anti-EGFR antibody was used as a model drug in this study. The binding of the antibody to EGFR overexpressed A431 cells was monitored in real time, which was found to follow the first-order kinetics with an association rate constant (ka) and dissociation rate constant (kd) of (2.7 ± 0.6) × 10(5) M(-1) s(-1) and (1.4 ± 0.5) × 10(-4) s(-1), respectively. The dissociation constant (KD) was determined to be 0.53 ± 0.26 nM with up to seven-fold variation among different individual A431 cells. In addition, the averaged A431 cell surface EGFR density was found to be 636/μm(2) with an estimation of 5 × 10(5) EGFR per cell. Additional measurement also revealed that different EGFR positive cell lines (A431, HeLa, and A549) show receptor density dependent anti-EGFR binding kinetics. The results demonstrate that SPRi is a valuable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resolution. Our findings show that the local environment affects the drug-receptor interactions, and in situ measurement of membrane protein binding kinetics is important.

The senses provide a means by which data on the physical and chemical properties of the environment may be collected and meaningfully interpreted. Sensation begins at the periphery, where a multitude of different sensory cell types are activated by environmental stimuli as different as photons and odorant molecules. Stimulus sensitivity is due to expression of different cell surface sensory receptors, and therefore the receptive field of each sense is defined by the aggregate of expressed receptors in each sensory tissue. Here, we review current understanding on patterns of expression and modes of regulation of sensory receptors. PMID:25938729

Construction aggregates, primarily stone, sand and gravel, are recovered from widespread naturally occurring mineral deposits and processed for use primarily in the construction industry. They are mined, crushed, sorted by size and sold loose or combined with portland cement or asphaltic cement to make concrete products to build roads, houses, buildings, and other structures. Much smaller quantities are used in agriculture, cement manufacture, chemical and metallurgical processes, glass production and many other products.

Part of the Annual Commodities Review 1995. Production of construction aggregates such as crushed stone and construction sand and gravel showed a marginal increase in 1995. Most of the 1995 increases were due to funding for highway construction work. The major areas of concern to the industry included issues relating to wetlands classification and the classification of crystalline silica as a probable human carcinogen. Despite this, an increase in demand is anticipated for 1996.

Summary Endoplasmic reticulum (ER) release and cell surface export of many G protein-coupled receptors (GPCRs), are tightly regulated. For GABAB receptors of GABA, the major mammalian inhibitory neurotransmitter, the ligand-binding GB1 subunit is maintained in the ER by unknown mechanisms in the absence of hetero-dimerization with the GB2 subunit. We report that GB1 retention is regulated by a specific gatekeeper, PRAF2. This ER resident transmembrane protein binds to GB1, preventing its progression in the biosynthetic pathway. GB1 release occurs upon competitive displacement from PRAF2 by GB2. PRAF2 concentration, relative to that of GB1 and GB2, tightly controls cell surfacereceptor density and controls GABAB function in neurons. Experimental perturbation of PRAF2 levels in vivo caused marked hyperactivity disorders in mice. These data reveal an unanticipated major impact of specific ER gate-keepers on GPCR function and identify PRAF2 as a new molecular target with therapeutic potential for psychiatric and neurological diseases involving GABAB function. PMID:26033241

Regulation and control of complex biological functions are dependent upon spatial organization of biological structures at many different length scales. For instance Eph receptors and their ephrin ligands bind when opposing cells come into contact during development, resulting in spatial organizational changes on the nanometer scale that lead to changes on the macro scale, in a process known as organ morphogenesis. One technique able to probe this important spatial organization at both the nanometer and micrometer length scales, including at cell-cell junctions, is stochastic optical reconstruction microscopy (STORM). STORM is a technique that localizes individual fluorophores based on the centroids of their point spread functions and then reconstructs a composite image to produce super resolved structure. We have applied STORM to study spatial organization of the cell surface of human breast cancer cells, specifically the organization of tyrosine kinase receptors and chemokine receptors. A better characterization of spatial organization of breast cancer cell surface proteins is necessary to fully understand the tumorigenisis pathways in the most common malignancy in United States women.

Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire. PMID:7964454

Aim: To investigate the effect of bleaching agents having a neutral pH on the surface of mineral trioxide aggregate (MTA) used as a coronal seal material for nonvital bleaching, beneath the bleaching agent, with the help of energy dispersive X-ray microanalysis and scanning electron microscopy (SEM). Materials and Methods: Six samples of plastic tubes filled with white MTA (Angelus white) were kept in 100% humidity for 21 days. Each sample was divided into 2 and made into 12 samples. These were then divided into three groups. Group A was exposed to Opalescence Boost 40% hydrogen peroxide (HP) (Ultradent). Group B to Opalescence 10% carbamide peroxide (Ultradent) and Group C (control group) not exposed to any bleaching agent. After recommended period of exposure to bleaching agents according to manufacturers’ instructions, the samples were observed under SEM with an energy dispersive X-ray microanalysis system (JSM-6380 LA). Results: There were no relevant changes in color and no statistically significant surface structure changes of the MTA in both the experimental groups. Conclusion: The present findings suggest that even high concentration HP containing bleaching agents with neutral pH can be used on the surface of MTA without causing structural changes. The superior sealing ability of MTA and the high alkalinity would prevent cervical resorption postbleaching. PMID:27656061

We previously reported that lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind NR5A nuclear receptors to regulate their activity. Here, the crystal structures of PIP2 and PIP3 bound to NR5A1 (SF-1) define a new interaction surface that is organized by the solvent-exposed PIPn headgroups. We find that stabilization by the PIP3 ligand propagates a signal that increases coactivator recruitment to SF-1, consistent with our earlier work showing that PIP3 increases SF-1 activity. This newly created surface harbors a cluster of human mutations that lead to endocrine disorders, thus explaining how these puzzling mutations cripple SF-1 activity. Finally, we propose that thismore » new surface acts as a PIP3-regulated interface between SF-1 and coregulatory proteins, analogous to the function of membrane-bound phosphoinositides.« less

Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR’s transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor’s ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR’s cytostatic action, the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Induction of p21Cip1 is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27Kip1 is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptorsurfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids. PMID:10373553

The delta opioid receptor (δR) is a promising alternate target for pain management, because δR agonists show decreased abuse potential compared to current opioid analgesics that target the mu opioid receptor. A critical limitation in developing δR as an analgesic target, however, is that δR agonists show relatively low efficacy in vivo, requiring the use of high doses that often cause adverse effects such as convulsions. Here we tested whether intracellular retention of δR in sensory neurons contributes to this low δR agonist efficacy in vivo by limiting surface δR expression. Using direct visualization of δR trafficking and localization, we define a phosphatase and tensin homolog (PTEN)-regulated checkpoint that retains δR in the Golgi and decreases surface delivery in rat and mice sensory neurons. PTEN inhibition releases δR from this checkpoint and stimulates delivery of exogenous and endogenous δR to the neuronal surface both in vitro and in vivo PTEN inhibition in vivo increases the percentage of TG neurons expressing δR on the surface, and allows efficient δR-mediated antihyperalgesia in mice. Together, we define a critical role for PTEN in regulating the surface delivery and bioavailability of the δR, explain the low efficacy of δR agonists in vivo, and provide evidence that active δR relocation is a viable strategy to increase δR antinociception.SIGNIFICANCE STATEMENTOpioid analgesics like morphine, which target the mu opioid receptor (μR), have been the mainstay of pain management, but their use is highly limited by adverse effects and their variable efficacy in chronic pain. Identifying alternate analgesic targets is therefore of great significance. While the delta opioid receptor (δR) is an attractive option, a critical limiting factor in developing δR as a target has been the low efficacy of δR agonists. Why δR agonists show low efficacy is still under debate. This study provides mechanistic and functional data that intracellular

The high affinity receptor for IgE, Fc epsilon receptor I (FcepsilonRI), is an activating immune receptor and key regulator of allergy. Antigen-mediated cross-linking of IgE-loaded FcepsilonRI alpha-chains induces cell activation via immunoreceptor tyrosine-based activation motifs in associated signaling subunits, such as FcepsilonRI gamma-chains. Here we show that the human FcepsilonRI alpha-chain can efficiently reach the cell surface by itself as an IgE-binding receptor in the absence of associated signaling subunits when the endogenous signal peptide is swapped for that of murine major histocompatibility complex class-I H2-K(b). This single-chain isoform of FcepsilonRI exited the endoplasmic reticulum (ER), trafficked to the Golgi and, subsequently, trafficked to the cell surface. Mutational analysis showed that the signal peptide regulates surface expression in concert with other described ER retention signals of FcepsilonRI-alpha. Once the FcepsilonRI alpha-chain reached the cell surface by itself, it formed a ligand-binding receptor that stabilized upon IgE contact. Independently of the FcepsilonRI gamma-chain, this single-chain FcepsilonRI was internalized after receptor cross-linking and trafficked into a LAMP-1-positive lysosomal compartment like multimeric FcepsilonRI. These data suggest that the single-chain isoform is capable of shuttling IgE-antigen complexes into antigen loading compartments, which plays an important physiologic role in the initiation of immune responses toward allergens. We propose that, in addition to cytosolic and transmembrane ER retention signals, the FcepsilonRI alpha-chain signal peptide contains a negative regulatory signal that prevents expression of an immunoreceptor tyrosine-based activation motif-free IgE receptor pool, which would fail to induce cell activation.

Cultured NIH-3T3 cells were transfected with cDNA constructs encoding human epidermal growth factor-receptor (EGF-R)* and two deletion mutants in the extracellular portion of the receptor molecule. One mutant is devoid of 124 amino-terminal amino acids, and the other lacks 76 residues. Mutant receptors were not delivered to the cell surface unless the transfected cells contained also endogenous EGF-Rs, suggesting that receptor interaction complements the mutation and allows surface display of mutant receptors. Immunoprecipitation experiments revealed an association between mutant and endogenous EGF-Rs when both proteins were expressed in the same cell. Hence, receptor-oligomers may exist in the plane of the membrane even in the absence of ligand binding, and oligomerization may play a role in normal trafficking of EGF-Rs to the cell surface. Mutant receptors retained partial ligand binding activity as 125I-labeled EGF was covalently cross-linked to both mutant receptors, and EGF stimulated, albeit weakly, their protein tyrosine kinase activity. Both mutant EGF-Rs bind EGF with a 10-fold lower affinity than that of the solubilized wild type EGF-R. These results provide further evidence that the region flanked by the two cysteine-rich domains plays a crucial role in defining ligand-binding specificity of EGF-R. Images PMID:2100196

To isolate collagen-binding cell surface proteins, detergent extracts of surface-iodinated MG-63 human osteosarcoma cells were chromatographed on affinity matrices of either type I collagen- Sepharose or Sepharose carrying a collagen-like triple-helical peptide. The peptide was designed to be triple helical and to contain the sequence Arg-Gly-Asp, which has been implicated as the cell attachment site of fibronectin, vitronectin, fibrinogen, and von Willebrand factor, and is also present in type I collagen. Three radioactive polypeptides having apparent molecular masses of 250 kD, 70 kD, and 30 kD were distinguishable in that they showed affinity toward the collagen and collagen-like peptide affinity columns, and could be specifically eluted from these columns with a solution of an Arg-Gly- Asp-containing peptide, Gly-Arg-Gly-Asp-Thr-Pro. These collagen-binding polypeptides associated with phosphatidylcholine liposomes, and the resulting liposomes bound specifically to type I collagen or the collagen-like peptide but not to fibronectin or vitronectin or heat- denatured collagen. The binding of these liposomes to type I collagen could be inhibited with the peptide Gly-Arg-Gly-Asp-Thr-Pro and with EDTA, but not with a variant peptide Gly-Arg-Gly-Glu-Ser-Pro. We conclude from these data that these three polypeptides are membrane molecules that behave as a cell surfacereceptor (or receptor complex) for type I collagen by interacting with it through the Arg-Gly-Asp tripeptide adhesion signal. The lack of binding to denatured collagen suggests that the conformation of the Arg-Gly-Asp sequence is important in the recognition of collagen by the receptor complex. PMID:3469204

Mixed color waste glass extracted from municipal solid waste is either not recycled, in which case it is an environmental and financial liability, or it is used in relatively low value applications such as normal weight aggregate. Here, we report on converting it into a novel glass-ceramic lightweight aggregate (LWA), potentially suitable for high added value applications in structural concrete (upcycling). The artificial LWA particles were formed by rapidly sintering (<10 min) waste glass powder with clay mixes using sodium silicate as binder and borate salt as flux. Composition and processing were optimized using response surface methodology (RSM) modeling, and specifically (i) a combined process-mixture dual RSM, and (ii) multiobjective optimization functions. The optimization considered raw materials and energy costs. Mineralogical and physical transformations occur during sintering and a cellular vesicular glass-ceramic composite microstructure is formed, with strong correlations existing between bloating/shrinkage during sintering, density and water adsorption/absorption. The diametrical expansion could be effectively modeled via the RSM and controlled to meet a wide range of specifications; here we optimized for LWA structural concrete. The optimally designed LWA is sintered in comparatively low temperatures (825-835 °C), thus potentially saving costs and lowering emissions; it had exceptionally low water adsorption/absorption (6.1-7.2% w/wd; optimization target: 1.5-7.5% w/wd); while remaining substantially lightweight (density: 1.24-1.28 g.cm(-3); target: 0.9-1.3 g.cm(-3)). This is a considerable advancement for designing effective environmentally friendly lightweight concrete constructions, and boosting resource efficiency of waste glass flows.

The growth of calcium oxalate monohydrate in the presence of Tamm-Horsfall protein (THP), osteopontin (OPN), and the 27-residue synthetic peptides (DDDS){sub 6}DDD and (DDDG){sub 6}DDD [where D = aspartic acid and X = S (serine) or G (glycine)] was investigated via in situ atomic force microscopy (AFM). The results show that these three growth modulators create extensive deposits on the crystal faces. Depending on the modulator and crystal face, these deposits can occur as discrete aggregates, filamentary structures, or uniform coatings. These proteinaceous films can lead to either the inhibition or increase of the step speeds (with respect to the impurity-free system) depending on a range of factors that include peptide or protein concentration, supersaturation and ionic strength. While THP and the linear peptides act, respectively, to exclusively increase and inhibit growth on the (-101) face, both exhibit dual functionality on the (010) face, inhibiting growth at low supersaturation or high modulator concentration and accelerating growth at high supersaturation or low modulator concentration. Based on analyses of growth morphologies and dependencies of step speeds on supersaturation and protein or peptide concentration, we argue for a picture of growth modulation that accounts for the observations in terms of the strength of binding to the surfaces and steps and the interplay of electrostatic and solvent-induced forces at crystal surface.

The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surfacereceptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus-receptor interaction crystallographically. Compared with extant HNV-G-ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus-host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure-function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations.

The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surfacereceptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus−receptor interaction crystallographically. Compared with extant HNV-G–ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus–host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure–function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations. PMID:25825759

The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV. PMID:1310996

Advanced glycosylation end-products (AGEs) are non-enzymatically glycosylated proteins that play an important role in several diseases and aging processes, including angiopathy, renal failure, diabetic complications, and some neurodegenerative diseases. In particular, glyceraldehyde (GCA)- and glycolaldehyde (GOA)-derived AGEs are deemed toxic AGEs, due to their cytotoxicity. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether or not macrophages recognize toxic AGEs through nucleolin receptors expressed on their surface. Free amino acid groups and arginine residues found in bovine serum albumin (BSA) were time-dependently modified by incubation with GCA and GOA. In addition, average molecular size was increased by incubation with GCA and GOA. While GCA-treated BSA (GCA-BSA) and GOA-treated BSA (GOA-BSA) were recognized by thioglycollate-elicited mouse peritoneal macrophages in proportion to their respective aldehyde-modification ratios, aldehyde-untreated control-BSA was not. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with GCA-BSA and GOA-BSA, but not with control-BSA. Further, pretreating macrophages with anti-nucleolin antibody, but not control-Immunoglobulin G, inhibited recognition of GCA-BSA and GOA-BSA by macrophages. Additionally, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited recognition of GCA-BSA and GOA-BSA. Moreover, nucleolin-transfected HEK293 cells recognized more GCA-BSA and GOA-BSA than control HEK cells did. Binding of nucleolin and GCA-BSA/GOA-BSA was also blocked by anti-nucleolin antibody at molecular level. These results indicate that nucleolin is a receptor that allows macrophages to recognize toxic AGEs.

We describe a method for nanoimaging interfacial dynamics and ligand-receptor binding at surfaces of live cells in 3-D. The imaging probe is a 1-μm diameter glass bead confined by a soft laser trap to create a "cloud" of fluctuating states. Using a facile on-line method of video image analysis, the probe displacements are reported at ~10 ms intervals with bare precisions (±SD) of 4-6 nm along the optical axis (elevation) and 2 nm in the transverse directions. We demonstrate how the Brownian distributions are analyzed to characterize the free energy potential of each small probe in 3-D taking into account the blur effect of its motions during CCD image capture. Then, using the approach to image interactions of a labeled probe with lamellae of leukocytic cells spreading on cover-glass substrates, we show that deformations of the soft distribution in probe elevations provide both a sensitive long-range sensor for defining the steric topography of a cell lamella and a fast telemetry for reporting rare events of probe binding with its surfacereceptors. Invoking established principles of Brownian physics and statistical thermodynamics, we describe an off-line method of super resolution that improves precision of probe separations from a non-reactive steric boundary to ~1 nm.

In the present paper, we have investigated the effect of ethanol in aqueous media, the pH and the presence of Ag nanoparticles (NPs) on the aggregation processes of the antitumoral anthraquinone parietin in aqueous media and on the metal surface. UV-visible absorption, fluorescence and Raman spectra of parietin were used for such purpose. The present study provides information about the deprotonation and molecular aggregation processes occurring in parietin under different environments: ethanol/water mixture and when adsorbed onto Ag nanoparticles. The effect of ethanol on the optical properties of parietin in alcohol-water mixtures was also investigated at different ethanol concentrations with the time. For the case of the adsorption and organization of parietin molecules on the surface of Ag NPs, special attention was paid to the use of surface-enhanced optical techniques, SEF (surface-enhanced fluorescence) and SERS (surface-enhanced Raman scattering), for the characterization of the parietin aggregates and the ionization of the molecule on the surface. In particular, we have studied the variation of the SEF signal with the pH, which depends on the molecular organization of the molecule on the surface. Furthermore, a detailed analysis of the SERS spectra at different pH was accomplished and the main Raman bands of the protonated, mono-deprotonated and di-deprotonated parietin were identified. Finally, the second ionization pK of parietin on metal NPs was deduced from the SERS spectra.

In the present paper, we have investigated the effect of ethanol in aqueous media, the pH and the presence of Ag nanoparticles (NPs) on the aggregation processes of the antitumoral anthraquinone parietin in aqueous media and on the metal surface. UV-visible absorption, fluorescence and Raman spectra of parietin were used for such purpose. The present study provides information about the deprotonation and molecular aggregation processes occurring in parietin under different environments: ethanol/water mixture and when adsorbed onto Ag nanoparticles. The effect of ethanol on the optical properties of parietin in alcohol-water mixtures was also investigated at different ethanol concentrations with the time. For the case of the adsorption and organization of parietin molecules on the surface of Ag NPs, special attention was paid to the use of surface-enhanced optical techniques, SEF (surface-enhanced fluorescence) and SERS (surface-enhanced Raman scattering), for the characterization of the parietin aggregates and the ionization of the molecule on the surface. In particular, we have studied the variation of the SEF signal with the pH, which depends on the molecular organization of the molecule on the surface. Furthermore, a detailed analysis of the SERS spectra at different pH was accomplished and the main Raman bands of the protonated, mono-deprotonated and di-deprotonated parietin were identified. Finally, the second ionization pK of parietin on metal NPs was deduced from the SERS spectra.

A deletion between amino acid residues Ser895 and Val1075 in the carboxyl terminus of the human calcium receptor (hCaR), which causes autosomal dominant hypocalcemia, showed enhanced signaling activity and increased cell surface expression in HEK293 cells (Lienhardt, A., Garabédian, M. G., Bai, M., Sinding, C., Zhang, Z., Lagarde, J. P., Boulesteix, J., Rigaud, M., Brown, E. M., and Kottler, M. L. (2000) J. Clin. Endocrinol. Metab. 85, 1695–1702). To identify the underlying mechanism(s) for these increases, we investigated the effects of carboxyl tail truncation and deletion in hCaR mutants using a combination of biochemical and cell imaging approaches to define motifs that participate in regulating cell surface numbers of this G protein-coupled receptor. Our data indicate a rapid constitutive receptor internalization of the cell surface hCaR, accumulating in early (Rab7 positive) and late endosomal (LAMP1 positive) sorting compartments, before targeting to lysosomes for degradation. Recycling of hCaR back to the cell surface was also evident. Truncation and deletion mapping defined a 51-amino acid sequence between residues 920 and 970 that is required for targeting to lysosomes and degradation but not for internalization or recycling of the receptor. No singular sequence motif was identified, instead the required sequence elements seem to distribute throughout this entire interval. This interval includes a high proportion of acidic and hydroxylated amino acid residues, suggesting a similarity to PEST-like degradation motif (PESTfind score of +10) and several glutamine repeats. The results define a novel large PEST-like sequence that participates in the sorting of internalized hCaR routed to the lysosomal/degradation pathway that regulates cell surfacereceptor numbers. PMID:22158862

The aggregation of virus particles is a particular form of colloidal self-assembly, since viruses of a give type are monodisperse and have identical, anisotropic surface charge distributions. In small-angle X-ray scattering experiments, the Qbeta virus was found to organize in different crystal structures in the presence of divalent salt and non-adsorbing polymer. Since a simple isotropic potential cannot explain the occurrence of all observed phases, we employ computer simulations to investigate how the surface charge distribution affects the virus interactions. Using a detailed model of the virus particle, we find an asymmetric ion distribution around the virus which gives rise to the different phases observed.

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators (RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide (EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins.

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surfacereceptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.

Supramolecular agglomerates of organic colorants based on noncovalent interactions are promising candidates for the development of sensors, optoelectronics, lighting, or photovoltaics. However, their fast and defect-free fabrication on large scales using low-cost technologies has proven elusive so far. Here, we introduce a so far unreported mechanism to induce molecular order in cyanine dyes within minutes from organic solvents by self-assembly. Spin coating blends of a cyanine dye and a soluble fullerene derivative ([6,6]-phenyl C(61)-butyric acid methyl ester (PCBM)) from apolar, aprotic solvents leads to phase-separated structures on the micrometer scale. With this superordinated phase structure, adjustment of dye aggregation is possible, leading to novel optical properties of the film emerging from dye self-assembly on the nanometer scale. In the primary process, semiporous PCBM domains act as nucleation sites for H-aggregates. H-aggregates can then be reconstructed into J-aggregates by dissolving PCBM from the film. Unexpectedly, the method even works for sterically hindered cyanine dyes that are known for their reduced tendency to aggregate. Additionally, selective removal of H-aggregates leaves a template of PCBM nanocrystals, onto which cyanine dye monomers readsorb from solution, forming H-aggregates of similar quality.

The Ca2+-sensing receptor (CaSR) is a member of family C of the GPCRs responsible for sensing extracellular Ca2+ ([Ca2+]o) levels, maintaining extracellular Ca2+ homeostasis, and transducing Ca2+ signaling from the extracellular milieu to the intracellular environment. In the present study, we have demonstrated a Ca2+-dependent, stoichiometric interaction between CaM and a CaM-binding domain (CaMBD) located within the C terminus of CaSR (residues 871–898). Our studies suggest a wrapping around 1–14-like mode of interaction that involves global conformational changes in both lobes of CaM with concomitant formation of a helical structure in the CaMBD. More importantly, the Ca2+-dependent association between CaM and the C terminus of CaSR is critical for maintaining proper responsiveness of intracellular Ca2+ responses to changes in extracellular Ca2+ and regulating cell surface expression of the receptor. PMID:20826781

Surface plasmon resonance (SPR) chips are widely used to measure association and dissociation rates for the binding kinetics between two species of chemicals, e.g., cell receptors and ligands. It is commonly assumed that ligands are spatially well mixed in the SPR region, and hence a mean-field rate equation description is appropriate. This approximation however ignores the spatial fluctuations as well as temporal correlations induced by multiple local rebinding events, which become prominent for slow diffusion rates and high binding affinities. We report detailed Monte Carlo simulations of ligand binding kinetics in an SPR cell subject to laminar flow. We extract the binding and dissociation rates by means of the techniques frequently employed in experimental analysis that are motivated by the mean-field approximation. We find major discrepancies in a wide parameter regime between the thus extracted rates and the known input simulation values. These results underscore the crucial quantitative importance of spatio-temporal correlations in binary reaction kinetics in SPR cell geometries, and demonstrate the failure of a mean-field analysis of SPR cells in the regime of high Damköhler number {{Da}}\\gt 0.1, where the spatio-temporal correlations due to diffusive transport and ligand-receptor rebinding events dominate the dynamics of SPR systems.

Class I major histocompatibility complex proteins appear to be the major cell surfacereceptors for simian virus 40 (SV40), as implied by the following observations. Adsorption of SV40 to LLC-MK2 rhesus monkey kidney cells specifically inhibited binding of a monoclonal antibody (MAb) against class I human lymphocyte antigen (HLA) proteins. Conversely, pretreatment of LLC-MK2 cells with anti-HLA MAbs inhibited infection by SV40. The ability of anti-HLA to inhibit infection was greatly reduced when the order of addition of the anti-HLA and the virus was reversed. Infection was also inhibited by preincubating SV40 with purified soluble class I protein. Finally, human lymphoblastoid cells of the Daudi line, which do not express class I major histocompatibility complex proteins, were infected at relatively low levels with SV40 virions. In a control experiment, we found that pretreatment of cells with a MAb specific for the leukocytic-function-associated antigen LFA-3 actually enhanced infection. This finding may also support the premise that class I major histocompatibility complex proteins are receptors for SV40.

Inhibitory neurons control the firing of glutamatergic neurons and synchronize brain activity. However, little is known about mechanisms of excitatory synapse formation in inhibitory neurons. Here we demonstrate that Erbin is specifically expressed in cortical inhibitory neurons. It localizes at excitatory synapses and regulates AMPA receptor (AMPAR) surface expression. Erbin mutation reduced mEPSCs and AMPAR currents specifically in parvalbumin (PV)-positive interneurons but not in pyramidal neurons. We found that the AMPAR auxiliary protein TARP γ-2 was specifically expressed in cortical interneurons. Erbin interacts with TARP γ-2 and is crucial for its stability. Deletion of the γ-2-interacting domain in Erbin attenuated surface AMPAR and excitatory transmission in PV-positive interneurons. Furthermore, we observed behavioral deficits in Erbin-null mice and in mice expressing an Erbin truncation mutant that is unable to interact with TARP γ-2. These observations demonstrate a crucial function for Erbin in AMPAR surface expression in cortical PV-positive interneurons and may contribute to a better understanding of psychiatric disorders.

The delta opioid receptor (DOPr), whilst not the primary target of clinically used opioids, is involved in development of opioid tolerance and addiction. There is growing evidence that DOPr trafficking is involved in drug addiction, e.g., a range of studies have shown increased plasma membrane DOPr insertion during chronic treatment with opioids. The present study used a transgenic mouse model in which the C-terminal of the DOPr is tagged with enhanced-green fluorescence protein to examine the effects of chronic morphine treatment on surface membrane expression in striatal cholinergic interneurons that are implicated in motivated learning following both chronic morphine and morphine sensitization treatment schedules in male mice. A sex difference was noted throughout the anterior striatum, which was most prominent in the nucleus accumbens core region. Incontrast with previous studies in other neurons, chronic exposure to a high dose of morphine for 6 days had no effect, or slightly decreased (anterior dorsolateral striatum) surface DOPr expression. A morphine sensitization schedule produced similar results with a significant decrease in surface DOPr expression in nucleus accumbens shell. These results suggest that chronic morphine and morphine sensitisation treatment may have effects on instrumental reward-seeking behaviours and learning processes related to drug addiction, via effects on striatal DOPr function.

Insulin resistance is a key driver of type 2 diabetes (T2D) and is characterized by defective insulin receptor (INSR) signalling. Although surface INSR downregulation is a well-established contributor to insulin resistance, the underlying molecular mechanisms remain obscure. Here we show that the E3 ubiquitin ligase MARCH1 impairs cellular insulin action by degrading cell surface INSR. Using a large-scale RNA interference screen, we identify MARCH1 as a negative regulator of INSR signalling. March1 loss-of-function enhances, and March1 overexpression impairs, hepatic insulin sensitivity in mice. MARCH1 ubiquitinates INSR to decrease cell surface INSR levels, but unlike other INSR ubiquitin ligases, MARCH1 acts in the basal state rather than after insulin stimulation. Thus, MARCH1 may help set the basal gain of insulin signalling. MARCH1 expression is increased in white adipose tissue of obese humans, suggesting that MARCH1 contributes to the pathophysiology of T2D and could be a new therapeutic target. PMID:27577745

The plasticity of excitatory synapses is an essential brain process involved in cognitive functions, and dysfunctions of such adaptations have been linked to psychiatric disorders such as depression. Although the intracellular cascades that are altered in models of depression and stress-related disorders have been under considerable scrutiny, the molecular interplay between antidepressants and glutamatergic signaling remains elusive. Using a combination of electrophysiological and single nanoparticle tracking approaches, we here report that the cognitive enhancer and antidepressant tianeptine (S 1574, [3-chloro-6-methyl-5,5-dioxo-6,11-dihydro-(c,f)-dibenzo-(1,2-thiazepine)-11-yl) amino]-7 heptanoic acid, sodium salt) favors synaptic plasticity in hippocampal neurons both under basal conditions and after acute stress. Strikingly, tianeptine rapidly reduces the surface diffusion of AMPA receptor (AMPAR) through a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent mechanism that enhances the binding of AMPAR auxiliary subunit stargazin with PSD-95. This prevents corticosterone-induced AMPAR surface dispersal and restores long-term potentiation of acutely stressed mice. Collectively, these data provide the first evidence that a therapeutically used drug targets the surface diffusion of AMPAR through a CaMKII-stargazin-PSD-95 pathway, to promote long-term synaptic plasticity.

Heating of protein- and sugar-containing materials is considered the primary factor affecting the formation of advanced glycation end products (AGEs). This study aimed to investigate the influence of heating conditions, digestion, and aggregation on the binding capacity of AGEs to the soluble AGE receptor (sRAGE). Samples consisting of mixtures of whey protein and lactose were heated at 130 °C. An in vitro infant digestion model was used to study the influence of heat treatment on the digestibility of whey proteins. The amount of sRAGE-binding ligands before and after digestion was measured by an ELISA-based sRAGE-binding assay. Water activity did not significantly affect the extent of digestibility of whey proteins dry heated at pH 5 (ranging from 3.3 ± 0.2 to 3.6 ± 0.1% for gastric digestion and from 53.5 ± 1.5 to 64.7 ± 1.1% for duodenal digestion), but there were differences in cleavage patterns of peptides among the samples heated at different pH values. Formation of sRAGE-binding ligands depended on the formation of aggregates and was limited in the samples heated at pH 5. Moreover, the sRAGE-binding activity of digested sample was changed by protease degradation and correlated with the digestibility of samples. In conclusion, generation of sRAGE-binding ligands during extensive heat treatment of whey protein/lactose mixtures is limited in acidic heating condition and dependent on glycation and aggregation.

The Melanocortin 5 Receptor (MC5R) is a cell surfacereceptor that belongs to the class of G-protein coupled receptors (GPCRs), which comprises an intracellular carboxylic domain, seven transmembrane helices and an extracellular amino terminal. Over the last few years, MC5R has been implicated in the regulation of lipid metabolism in exocrine glands, muscle and even in adipose tissue and its function is quite dependent on its correct cell membrane targeting. In this context, the purpose of this work was to study the role of MC5R N-terminus in the receptor trafficking from the Endoplasmic Reticulum (ER) through the Golgi complex to the plasma membrane. Analysis of N-terminal deleted forms of MC5R revealed that the first 21 amino acids contain the information responsible for the receptor cell surface expression and the removal of further amino acids interfere with the receptor synthesis. In this setting, several mutant forms of the receptor were created by site directed mutagenesis of the MC5R first 21 amino acids and their presence at the plasma membrane was assessed. We have found that two small motifs, constituted by residues Ser4/Ser5 and Ser17/Glu18, are clearly involved in the correct targeting of MC5R to the cell surface. Fluorescence microscopy analysis have revealed that MC5R constructs with mutations in those residues are mainly retained at the ER/Golgi complex. Furthermore, the homodimerization ability of the receptor is maintained in these mutant forms, suggesting that other mechanisms are involved in the regulation of the anterograde transport of MC5R by those N-terminal domains.

Corticotropin releasing factor binding protein (CRF-BP) was originally recognized as CRF sequestering protein. However, its differential subcellular localization in different brain nuclei suggests that CRF-BP may have additional functions. There is evidence that CRF-BP potentiates CRF and urocortin 1 actions through CRF type 2 receptors (CRF2R). CRF2R is a G protein-coupled receptor (GPCR) that is found mainly intracellularly as most GPCRs. The access of GPCRs to the cell surface is tightly regulated by escort proteins. We hypothesized that CRF-BP binds to CRF2R, exerting an escort protein role. We analyzed the colocalization of CRF-BP and CRF2R in cultured rat mesencephalic neurons, and the localization and interaction of heterologous expressed CRF-BP and CRF2αR in yeast, human embryonic kidney 293, and rat pheochromocytoma 12 cells. Our results showed that CRF-BP and CRF2R naturally colocalize in the neurites of cultured mesencephalic neurons. Heterologous expression of each protein showed that CRF-BP was localized mainly in secretory granules and CRF2αR in the endoplasmic reticulum. In contrast, CRF-BP and CRF2αR colocalized when both proteins are coexpressed. Here we show that CRF-BP physically interacts with the CRF2αR but not the CRF2βR isoform, increasing CRF2αR on the cell surface. Thus, CRF-BP emerges as a GPCR escort protein increasing the understanding of GPCR trafficking. PMID:27035969

Corticotropin releasing factor binding protein (CRF-BP) was originally recognized as CRF sequestering protein. However, its differential subcellular localization in different brain nuclei suggests that CRF-BP may have additional functions. There is evidence that CRF-BP potentiates CRF and urocortin 1 actions through CRF type 2 receptors (CRF2R). CRF2R is a G protein-coupled receptor (GPCR) that is found mainly intracellularly as most GPCRs. The access of GPCRs to the cell surface is tightly regulated by escort proteins. We hypothesized that CRF-BP binds to CRF2R, exerting an escort protein role. We analyzed the colocalization of CRF-BP and CRF2R in cultured rat mesencephalic neurons, and the localization and interaction of heterologous expressed CRF-BP and CRF2αR in yeast, human embryonic kidney 293, and rat pheochromocytoma 12 cells. Our results showed that CRF-BP and CRF2R naturally colocalize in the neurites of cultured mesencephalic neurons. Heterologous expression of each protein showed that CRF-BP was localized mainly in secretory granules and CRF2αR in the endoplasmic reticulum. In contrast, CRF-BP and CRF2αR colocalized when both proteins are coexpressed. Here we show that CRF-BP physically interacts with the CRF2αR but not the CRF2βR isoform, increasing CRF2αR on the cell surface. Thus, CRF-BP emerges as a GPCR escort protein increasing the understanding of GPCR trafficking.

Platelet spreading and retraction play a pivotal role in the platelet plugging and the thrombus formation. In routine laboratory, platelet function tests include exhaustive information about the role of the different receptors present at the platelet surface without information on the 3D structure of platelet aggregates. In this work, we develop, a method in Digital Holographic Microscopy (DHM) to characterize the platelet and aggregate 3D shapes using the quantitative phase contrast imaging. This novel method is suited to the study of platelets physiology in clinical practice as well as the development of new drugs. PMID:26417523

MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl(/sup 3/H)tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl(/sup 3/H)tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface.

and the horizontal advective scale. The advective scale, refers to how advective processes modify scalar fluxes of a given patch by its surrounding patches. The convective scales relates to processes that distort the equilibrium between the ABL and the underlying surface. In this study, I focus on the advective case, comparing the heterogeneity length scale to the downwind distance required by the local boundary layer to equilibrate with the surface (i.e. the statistics become virtually 1-dimensional). In order to do so, numerical simulations, using a Reynolds average closure models of the flow over a discontinuity on vegetation cover are performed aiming to establish the distance from the discontinuity at which the equilibrium with the new surface is re-establish. Comparing this length with a characteristic length scale of the heterogeneity will provide a framework to quantify uncertainties associated with regional exchange estimates from flux aggregation over heterogeneous landscape.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis and preferentially kills tumor cells by engaging specific glycosylated death receptors, resulting in the internalization of ligand/receptor complexes and recruitment of the initiator caspase-8 to an activation platform known as the death-inducing signaling complex (DISC). However, emergence of TRAIL-resistant sub-populations may contribute to therapeutic failure. To investigate resistance mechanisms, we isolated a stable TRAIL-resistant sub-population of the metastatic colon cancer cell line LS-LIM6, designated LIM6-TR. LIM6-TR cells are impaired in endocytosis of TRAIL/death receptors complexes and failed to recruit/activate caspase-8 to the DISC upon TRAIL stimulation. Differential activation of Wnt and JNK pathways is not responsible for acquisition of TRAIL resistance. LIM6-TR cells display a marked increase in cell-surface expression of galectin-3, an endogenous lectin, which co-localizes with and binds death receptors. Silencing of galectin-3 restores TRAIL sensitivity and promotes TRAIL-mediated endocytosis of TRAIL/death receptors complexes. Inhibitors of galectin-3 and glycosylation also re-sensitize LIM6-TR to TRAIL and restore internalization of ligand/receptors complexes. These studies identify a novel TRAIL-resistance mechanism in which galectin-3 impedes trafficking of death receptor by anchoring them in glycan nano-clusters, blocking the execution of the apoptosis signal.

The present study was undertaken to determine the relationship between the hyaluronate receptor and CD44 (H-CAM), cell-surface glycoproteins of similar molecular weights that have been implicated in cell adhesion. In initial experiments, a panel of monoclonal antibodies directed against CD44 were tested for their ability to cross react with the hyaluronate receptor. These antibodies immunoprecipitated [3H]hyaluronate binding activity from detergent extracts of both mouse and human cells, indicating that the hyaluronate receptor is identical to CD44. In addition, one of these antibodies (KM-201 to mouse CD44) directly blocked the binding of labeled hyaluronate to the receptor and inhibited hyaluronate dependent aggregation of SV-3T3 cells. CD44 has also been implicated in lymphocyte binding to high endothelial venules during lymphocyte homing. Interestingly, the monoclonal antibody Hermes- 3, which blocks lymphocyte binding to the high endothelial venules of mucosal lymphoid tissue, had no effect on the binding of labeled hyaluronate. Furthermore, the binding of lymphocytes to high endothelial cells of lymph nodes and mucosal lymphoid tissue was not significantly affected by treatment with agents that block the binding of hyaluronate (hyaluronidase, excess hyaluronate and specific antibodies). Thus, CD44 appears to have at least two distinct functional domains, one for binding hyaluronate and another involved in interactions with mucosal high endothelial venules. PMID:1703543

Nanoparticle electrodeposition is a simple and scalable approach to synthesizing supported nanoparticles. Used with a deep eutectic solvent (DES), surface nanoparticles can be assembled and exhibit unique surface charge separation when the DES is adsorbed on the nanoparticle surface. Key to understanding and controlling the assembly and the capacitance is a thorough understanding of surface particle mobility and charge screening, which requires an in-situ approach. In this study, Pb particle formation, size, shape and capacitance are resolved in a 1:2 choline Cl–: urea deep eutectic solvent whilst sweeping the cell potential in the range: 0.2 V to –1.2 V (vs.more » Ag/AgCl). These system parameters were resolved using a complementary suite of sample-rotated small angle X-ray scattering (SR-SAXS) and electrochemical impedance spectroscopy (EIS), which are presented and discussed in detail. This approach is able to show that both particle and ion transport are impeded in the DES, as aggregation occurs over the course of 6 minutes, and dissolved Pb ions accumulate and remain near the surface after a nucleation pulse is applied. The DES-Pb interactions strongly depend on the cell potential as evidenced by the specific differential capacitance of the Pb deposit, which has a maximum value of 2.5 +/– 0.5 F g–1 at –1.0 V vs. Ag/AgCl. Together, the SR-SAXS-EIS approach is able to characterize the unique nanoparticle capacitance, mobility and ion mobility in a DES and can be used to study a wide range of nanoparticle deposition systems in-situ.« less

1. The aggregation of acetylcholine receptors at the developing neuromuscular junction is critical to the development and function of this synapse. In vitro studies have shown that receptoraggregation can be induced by the finding of agrin to the muscle cell surface and by the electric field-induced concentration of a (nonreceptor) molecule at the cathodal cell pole. 2. We report here on the interaction between agrin binding and electric fields with respect to the distribution of receptors and agrin binding sites. 3. (a) Pretreatment of cells with agrin completely blocks the development of field-induced receptor clusters. (b) Field-induced aggregation of receptors precedes the field-induced aggregation of agrin binding sites by approximately 30 min. (c) Electric fields prevent agrin-induced receptor clustering despite the presence of agrin binding sites and freely diffusing receptors. 4. These results indicate that another membrane component-but not the agrin binding site and not the receptor-is required for agrin-induced receptor clustering. They also suggest that electric fields and agrin cause receptor clustering via common molecular mechanisms.

Antibodies able to bind and modify the function of cell surface signaling components in vivo are increasingly being used as therapeutic drugs. The identification of such "functional" antibodies from within large antibody pools is, therefore, the subject of intense research. Here we describe a novel cell-based expression and reporting system for the identification of functional antibodies from antigen-binding populations preselected with phage display. The system involves inducible expression of the antibody gene population from the Rosa-26 locus of embryonic stem (ES) cells, followed by secretion of the antibodies during ES cell differentiation. Target antigens are cell-surface signaling components (receptors or ligands) with a known effect on the direction of cell differentiation (FGFR1 mediating ES cell exit from self renewal in this particular protocol). Therefore, inhibition or activation of these components by functional antibodies in a few elite clones causes a shift in the differentiation outcomes of these clones, leading to their phenotypic selection. Functional antibody genes are then recovered from positive clones and used to produce the purified antibodies, which can be tested for their ability to affect cell fates exogenously. Identified functional antibody genes can be further introduced in different stem cell types. Inducible expression of functional antibodies has a temporally controlled protein-knockdown capability, which can be used to study the unknown role of the signaling pathway in different developmental contexts. Moreover, it provides a means for control of stem cell differentiation with potential in vivo applications.

Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.

Macropinocytosis is an ancient mechanism that allows cells to harvest nutrients from extracellular media, which also allows immune cells to sample antigens from their surroundings. During macropinosome formation, bulk plasma membrane is internalized with all its integral proteins. It is vital for cells to salvage these proteins before degradation, but the mechanisms for sorting them are not known. Here we describe the evolutionarily conserved recruitment of the WASH (WASP and SCAR homolog) complex to both macropinosomes and phagosomes within a minute of internalization. Using Dictyostelium, we demonstrate that WASH drives protein sorting and recycling from macropinosomes and is thus essential to maintain surfacereceptor levels and sustain phagocytosis. WASH functionally interacts with the retromer complex at both early and late phases of macropinosome maturation, but mediates recycling via retromer-dependent and -independent pathways. WASH mutants consequently have decreased membrane levels of integrins and other surface proteins. This study reveals an important pathway enabling cells to sustain macropinocytosis without bulk degradation of plasma membrane components. PMID:27647881

Cyclosporin A (CsA) is an immunosuppressive agent that inhibits the synthesis of lymphokines by T lymphocytes at the level of transcription. A cytoplasmic protein, cyclophilin, is the most thoroughly studied CsA-binding protein, but its ubiquitous presence in cells of all types raises questions about its role in immunosuppression. In an attempt to ascertain the presence of a cell surfacereceptor, we synthesized two polyvalent macromolecular CsA derivatives, CsA-BBa-ovalbumin and CsA-BBa-aminodextran (CBD), from the product of the photochemical reaction of CsA and 4-benzoylbenzoic acid (CsA-BBa). (i) They inhibited the peptidylprolyl cis-trans isomerase activity of cyclophilin and the synthesis of interleukin 2 by phorbol ester-activated EL-4 cells. (ii) CBD also inhibited interleukin 2 secretion by Con A-activated T-cell-enriched mouse splenocytes. 4-Benzoylbenzoic acid (BBa)-aminodextran and aminodextran were inactive. (iii) Direct binding and competition studies with [3H]CsA indicated that CBD does not enter EL-4 cells (i.e., it acted at the surface). (iv) CBD caused agglutination of EL-4 cells, murine B and T lymphocytes, human thymocytes, and two T-cell hybridomas. Agglutination was inhibited by a monoclonal antibody to CsA and by CsA and CsA-BBa, but not by BBa. No agglutination was seen with BBa-aminodextran or aminodextran. HeLa cells, Vero (monkey kidney) cells, a mouse plasmacytoma, COS cells, and a poorly differentiated B-cell lymphoma were not agglutinated. (v) EL-4 cells failed to be agglutinated after treatment with trypsin or chymotrypsin. Specific agglutination was again possible after incubation for 5 h at 37 degrees C in the absence of enzyme. (vi) CBD covalently linked to crosslinked agarose beads inhibited interleukin 2 production by phorbol ester-stimulated EL-4 cells. No activity was seen if cell-to-bead contact was prevented by a 0.02-microns microporous filter that did not interfere with the passage of CBD. Our findings support the presence

Infection by the Human immunodeficiency virus (HIV) can result in debilitating neurological syndromes collectively known as HIV associated neurocognitive disorders (HAND). While the HIV coat protein gp120 has been identified as a potent neurotoxin that enhances NMDA receptor function, the exact mechanisms for effect are not known. Here we provide evidence that gp120 activates two separate signaling pathways that converge to enhance NMDA-evoked calcium flux by clustering NMDA receptors in modified membrane microdomains. HIV gp120 enlarged, and stabilized the structure of lipid rafts on neuronal dendrites by mechanisms that involved a redox-regulated translocation of a sphingomyelin hydrolase (neutral sphingomyelinase-2; nSMase2) to the plasma membrane. A concurrent pathway was activated that enhanced the forward traffic of NMDA receptors by promoting a PKA-dependent phopshorylation of the NR1 C-terminal serine 897 (that masks an ER retention signal), followed by a PKC-dependent phosphorylation of serine 896 (important for surface expression). NMDA receptors were preferentially targeted to synapses, and clustered in modified membrane microdomains. In these conditions, NMDA receptors were unable to laterally disperse, and did not internalize, even in response to strong agonist induction. Focal NMDA-evoked calcium bursts were enhanced three-fold in these regions. Inhibiting membrane modification or NR1 phosphorylation prevented gp120 from enhancing the surface localization and clustering of NMDA receptors, while disrupting the structure of membrane microdomains restored the ability of NMDA receptors to disperse and internalize following gp120. These findings demonstrate that gp120 contributes to synaptic dysfunction in the setting of HIV-infection by interfering with the traffic of NMDA receptors. PMID:22114277

Background The importance of surface epithelium and epithelial inclusion cysts in the ovary arises from studies demonstrating that these structures are susceptible to epithelial ovarian cancer development. The expression of estrogen receptor alpha (ER alpha), androgen receptor (AR), in epithelial cells of the ovary from premenopausal and postmenopausal women is interesting because sexual steroid hormones are involved in cell growth and differentiation. Methods The presence of ER alpha, AR, and the orphan G protein-coupled receptor 30 (GPR30) was demonstrated by immunofluorescence in ovaries obtained from 79 pre and postmenopausal patients, undergoing histero-salpingo-oophorectomy for proliferative gynecological diseases. The proportion of patients that displayed positive reaction for estrogen and androgen receptors in epithelial cells of the ovary was evaluated according to menopausal status and associated pathology. Results The proportion of patients that displayed a positive receptor expression in the epithelial cells of the ovarian surface and cortical inclusion cysts shows that ER alpha is present in 20 of 79 patients (0.25), AR in 33 of 79 (0.42) and GPR30 in 38 of 55 (0.69). There are no differences in ER alpha, AR, and GPR30 expression between pre and postmenopausal patients and considering the associated pathology, proportions for ER alpha and GPR30 are similar. The patients with cervical cancer show a higher proportion of AR expression in epithelial cells of the ovary, which is statistically significant (P surface epithelial ovarian cells and its derivatives are observed with a proportion that is specific for each receptor. The proportion of expression for these receptors in the epithelial cells of the ovary does not change after menopause. The proportion of ovaries with AR positive epithelial cells in patients with cervical

This in vitro study evaluates the morphological changes of the root surface and fracture resistance after treatment of root cracks by CO2 laser and glass Ionomer or mineral trioxide aggregates (MTA). Fifty freshly extracted human maxillary central incisor teeth with similar dimension were selected. Crowns were sectioned at the cemento-enamel junction, and the lengths of the roots were adjusted to 13 mm. A longitudinal groove with a dimension of 1×5 mm2 and a depth of 1.5 mm was prepared by a high speed fissure bur on the labial surface of the root. The roots were divided into 5 groups: the 10 root grooves in group 1 were remained unfilled and were used as a control group. The 10 root grooves in group 2 were filled with glass Ionomer, 10 root grooves in group 3 were filled with MTA, the 10 root grooves in group 4 were filled with glass Ionomer and irradiated by CO2 laser and the 10 root grooves in group 5 were filled with MTA and irradiated with CO2 laser. Scanning electron microscopy was performed for two samples in each group. Tests for fracture strength were performed using a universal testing machine and a round tip of a diameter of 4 mm. The force was applied vertically with a constant speed of 1 mm min 1. For each root, the force at the time of fracture was recorded in Newtons. Results were evaluated statistically with ANOVA and Turkey's Honestly Significant Difference (HSD) tests. SEM micrographs revealed that the melted masses and the plate-like crystals formed a tight Chemical bond between the cementum and glass Ionomer and melted masses and globular like structure between cementum and MTA. The mean fracture resistance was the maximum fracture resistance in group 5 (810.8 N). Glass Ionomer and MTA with the help of CO2 laser can be an alternative to the treatment of tooth crack or fracture. CO2 laser increase the resistance of the teeth to fracture.

Extracellular nucleotides transmit signals into the cells through the P2 family of cell surfacereceptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y2 receptor (P2Y2R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-β-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1–10 μm UTP, a selective P2Y2R agonist, displaced the P2Y2R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y2R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y2R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y2R in intact human blood vessels. PMID:19996104

The structural simplicity of native cyclodextrins (CDs) contrasts with their complex behavior in the bulk of aqueous solutions, mainly when they are combined with other cosolutes. Many scientific and industrial applications based on these molecules are supported only by empirical information. The lack of fundamental knowledge, which would allow one to rationally optimize many of these applications, is notable mainly at the solution/air interface. Basic information on phenomena such as the spontaneous adsorption of native CDs or on the structure of CD aggregates in the bulk solution is really scarce. In order to fill these gaps, a detailed computational study on the adsorption and aggregation of α- and β-CDs as a function of temperature is presented here. Our simulations reproduce, at atomic resolution, the experimentally observed much higher ability of β-CD to aggregate compared to that of α-CD at 298 K, as well as their dependence on temperature. The adsorption of both individual CDs and small CD aggregates (up to 20 molecules) to the solution/air interface is found to be negligible. 0.8 μs long trajectories of single CD molecules in aqueous solution reveal that the main differences in the behavior of both CDs are their flexibility, higher for β-CD, and the occupancy of individual intramolecular hydrogen bonds that is significantly longer for the same cyclodextrin. The aggregation pattern of α- and β-CDs is followed at the hundreds of ns time scale, allowing both the spontaneous self-assembly of cyclodextrins and their redistribution along the aggregates to be observed. This is the first attempt to study the adsorption and aggregation of native cyclodextrins by atomistic molecular dynamics simulations.

A chronic intermittent ethanol (CIE) exposure regimen consists of repeated episodes of ethanol intoxication and withdrawal. CIE treatment has been reported to result in a significant enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in vivo, and trafficking of NMDA receptors is emerging a key regulatory mechanism that underlies the channel function. Therefore, in the present study, we examined the effects of CIE on NMDA receptor subunit surface expression. Cultured cortical neurons were exposed to 75 mM ethanol for 14 h followed by 10 h of withdrawal, repeated this cycle five times, and followed by 2 or 5 days of withdrawal. Surface-expressed NMDA receptor subunits and their endocytosis were measured by biotinylation and Western blots. CIE significantly increased NMDA receptor (NR) 1 and NR2B but not NR2A subunit surface expression after 5 days of treatment. However, CIE treatment did not reduce the NMDA receptor endocytosis. Quantification of immunocytochemistry confirmed CIE-induced increase in both the total number of NR1 and NR2B subunit clusters and their targeting to synaptic sites. It is noteworthy that this effect persisted even after ethanol withdrawal with a peak expression occurring between 0 and 2 days after withdrawal, and the expression on the plasma membrane was still at high levels after 5 days of withdrawal. In addition, this was accompanied by significant increases in postsynaptic density protein 95 clusters. Protein kinase A inhibitor completely reversed CIE-induced increase in NR1 and partially in NR2B surface level and a long-lasting effect. These changes may contribute to the development of ethanol-induced neurotoxicity and ethanol dependence.

The murine cytomegalovirus-encoded protein m157 is a cognate ligand for both inhibitory and activating receptors expressed by natural killer cells. Additionally, m157 is expressed on the surface of infected cells by a glycophosphatidylinositol (GPI) anchor. Although endogenous GPI-anchored proteins are known to be ligands for the NK cell receptor, NKG2D, the contribution of the GPI anchor for viral m157 ligand function is unknown. To determine whether the GPI anchor for m157 is dispensable for m157 function, we generated m157 variants expressed as transmembrane fusion proteins and tested cells expressing transmembrane m157 for the capacity to activate cognate Ly49 receptors. We found that the GPI anchor is required for high-level cell surface expression of m157, and that the transmembrane m157 ligand retains the capacity to activate reporter cells and NK cells expressing Ly49H, as well as Ly49I129 reporter cells, but with reduced potency. Importantly, target cells expressing the transmembrane form of m157 were killed less efficiently and failed to mediate Ly49H receptor downregulation on fresh NK cells compared to targets expressing GPI-anchored m157. Taken together, these results show that the GPI anchor for m157 facilitates robust cell surface expression, and that NK cells are sensitive to the altered cell surface expression of this potent viral evasin. PMID:23840655

The physiological functions of glycine receptors (GlyRs) depend on their subcellular locations. In axonal terminals of the central neurons, GlyRs trigger a slow facilitation of presynaptic transmitter release; however, their spatial relationship to the release sites is not known. In this study, we examined the distribution of GlyRs in the rat glutamatergic calyx of Held nerve terminal using high-resolution pre-embedding immunoelectron microscopy. We performed a quantitative analysis of GlyR-associated immunogold (IG) labeling in 3D reconstructed calyceal segments. A variable density of IG particles and their putative accumulations, inferred from the frequency distribution of inter-IG distances, indicated a non-uniform distribution of the receptors in the calyx. Subsequently, increased densities of IG particles were found in calyceal swellings, structures characterized by extensive exocytosis of glutamate. In swellings as well as in larger calyceal stalks, IG particles did not tend to accumulate near the glutamate releasing zones. On the other hand, GlyRs in swellings (but not in stalks) preferentially occupied membrane regions, unconnected to postsynaptic cells and presumably accessible by ambient glycine. Furthermore, the sites with increased GlyR concentrations were found in swellings tightly juxtaposed with GABA/glycinergic nerve endings. Thus, the results support the concept of an indirect mechanism underlying the modulatory effects of calyceal GlyRs, activated by glycine spillover. We also suggest the existence of an activity-dependent mechanism regulating the surface distribution of α homomeric GlyRs in axonal terminals of central neurons. PMID:25339867

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death. PMID:19887445

In the kidney the epithelial Na+ channel (ENaC) is co-expressed with the sulfonylurea receptor (SUR), an ABC protein that shares a high degree of homology with the cystic fibrosis transmembrane conductance regulator (CFTR) and reportedly modifies ENaC in various preparations. To investigate a possible regulatory relationship between SUR and ENaC, we performed co-expression studies on Xenopus laevis oocytes, which were assayed for amiloride-sensitive currents (DeltaIami). Moreover, a chemiluminescence assay was used to investigate the surface expression of extracellular hemagglutinin-tagged SUR1 (SUR1-HA) or HA-tagged ENaC (ENaC-HA). In oocytes co-injected with SUR1/ENaC (or SUR2B/ENaC) DeltaIami was reduced by congruent with 53% (or congruent with 45%) compared to DeltaIami measured in matched control oocytes injected with ENaC alone. The inhibitory effect of SUR on DeltaIami was preserved in oocytes expressing ENaC with C-terminally truncated subunits. Co-expression of SURs did not confer sensitivity of DeltaIami to diazoxide, pinacidil, tolbutamide, or glibenclamide. ENaC does not facilitate the surface expression of SUR1-HA, which is known to be retained in the endoplasmatic reticulum (ER) by an ER-retention/retrieval signal. SUR1-HAAAA, a mutant that lacks this signal, still inhibits ENaC currents. Chemiluminescence was reduced by congruent with 49% in oocytes co-expressing ENaC-HA/SUR1 compared to that in control oocytes expressing ENaC-HA alone. We conclude that SUR does not interact with ENaC at the level of the plasma membrane but that it inhibits DeltaIami by reducing surface expression of the channel.

N-methyl-d-aspartate receptors (NMDARs) are ion channels whose synaptic versus extrasynaptic localization critically influences their functions. This distribution of NMDARs is highly dependent on their lateral diffusion at the cell membrane. Each obligatory subunit of NMDARs (GluN1 and GluN2) contains two extracellular clamshell-like domains with an agonist-binding domain and a distal N-terminal domain (NTD). To date, the roles and dynamics of the NTD of the GluN1 subunit in NMDAR allosteric signaling remain poorly understood. Using single nanoparticle tracking in mouse neurons, we demonstrate that the extracellular neuronal protease tissue-type plasminogen activator (tPA), well known to have a role in the synaptic plasticity and neuronal survival, leads to a selective increase of the surface dynamics and subsequent diffusion of extrasynaptic NMDARs. This process explains the previously reported ability of tPA to promote NMDAR-mediated calcium influx. In parallel, we developed a monoclonal antibody capable of specifically blocking the interaction of tPA with the NTD of the GluN1 subunit of NMDAR. Using this original approach, we demonstrate that the tPA binds the NTD of the GluN1 subunit at a lysine in position 178. Accordingly, when applied to mouse neurons, our selected antibody (named Glunomab) leads to a selective reduction of the tPA-mediated surface dynamics of extrasynaptic NMDARs, subsequent signaling and neurotoxicity, both in vitro and in vivo. Altogether, we demonstrate that the tPA is a ligand of the NTD of the obligatory GluN1 subunit of NMDAR acting as a modulator of their dynamic distribution at the neuronal surface and subsequent signaling. PMID:27831563

The location and density of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is controlled by scaffolding proteins within the postsynaptic density (PSD). SAP97 is a PSD protein with two N-terminal isoforms, α and β, that have opposing effects on synaptic strength thought to result from differential targeting of AMPA receptors into distinct synaptic versus extrasynaptic locations, respectively. In this study, we have applied dSTORM super resolution imaging in order to localize the synaptic and extrasynaptic pools of AMPA receptors in neurons expressing α or βSAP97. Unexpectedly, we observed that both α and βSAP97 enhanced the localization of AMPA receptors at synapses. However, this occurred via different mechanisms: αSAP97 increased PSD size and consequently the number of receptor binding sites, whilst βSAP97 increased synaptic receptor cluster size and surface AMPA receptor density at the PSD edge and surrounding perisynaptic sites without changing PSD size. αSAP97 also strongly enlarged presynaptic active zone protein clusters, consistent with both presynaptic and postsynaptic enhancement underlying the previously observed αSAP97-induced increase in AMPA receptor-mediated currents. In contrast, βSAP97-expressing neurons increased the proportion of immature filopodia that express higher levels of AMPA receptors, decreased the number of functional presynaptic terminals, and also reduced the size of the dendritic tree and delayed the maturation of mushroom spines. Our data reveal that SAP97 isoforms can specifically regulate surface AMPA receptor nanodomain clusters, with βSAP97 increasing extrasynaptic receptor domains at peri-synaptic and filopodial sites. Moreover, βSAP97 negatively regulates synaptic maturation both structurally and functionally. These data support diverging presynaptic and postsynaptic roles of SAP97 N-terminal isoforms in synapse maturation and plasticity. As numerous splice isoforms exist in

ABSTRACT In traditional models of in vitro biofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development of Pseudomonas aeruginosa biofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. PMID:27006463

The field of making, studying and using molecular aggregates, in which the individual molecules (monomers) are arranged in a regular fashion, has come a long way. Taking control over the aggregation of small molecules and polymers in bulk, on surfaces and at interfaces pose a considerable challenge for their utilization in modern high tech applications. In this review we provide a detailed insight into recent trends in molecular aggregates from the perspectives of nanoarchitectonics.

Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell

The molecular mechanisms that control the targeting of newly synthesized G protein-coupled receptors (GPCRs) to the functional destinations remain poorly elucidated. Here, we have determined the role of Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins 1 and 2 (GGA1 and GGA2) in the cell surface transport of α2B-adrenergic receptor (α2B-AR), a prototypic GPCR, and studied the underlying mechanisms. We demonstrated that knockdown of GGA1 and GGA2 by shRNA and siRNA significantly reduced the cell surface expression of inducibly expressed α2B-AR and arrested the receptor in the perinuclear region. Knockdown of each GGA markedly inhibited the dendritic expression of α2B-AR in primary cortical neurons. Consistently, depleting GGA1 and GGA2 attenuated receptor-mediated signal transduction measured as ERK1/2 activation and cAMP inhibition. Although full length α2B-AR associated with GGA2 but not GGA1, its third intracellular loop was found to directly interact with both GGA1 and GGA2. More interestingly, further mapping of interaction domains showed that the GGA1 hinge region and the GGA2 GAE domain bound to multiple subdomains of the loop. These studies have identified an important function and revealed novel mechanisms of the GGA family proteins in the forward trafficking of a cell surface GPCR. PMID:27901063

Abstract There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. We have quantified the spatiotemporal dynamics of the redistribution of immunoglobulin E-loaded receptors (IgE-FcɛRI) on rat basophilic leukemia-2H3 mast cells in contact with fluid and gel-phase membranes displaying ligands for immunoglobulin E, using total internal reflection fluorescence microscopy. To clearly separate the kinetics of receptor redistribution from cell spreading, and to precisely define the initial contact time (±50 ms), micropipette cell manipulation was used to bring individual cells into contact with surfaces. On ligand-free surfaces, there are micron-scale heterogeneities in fluorescence that likely reflect regions of the cell that are more closely apposed to the substrate. When ligands are present, receptor clusters form with this same size scale. The initial rate of accumulation of receptors into the clusters is consistent with diffusion-limited trapping with D ∼10−1μm2/s. These results support the hypothesis that clusters form by diffusion to cell-surface contact regions. Over longer timescales (>10 s), individual clusters moved with both diffusive and directed motion components. The dynamics of the cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes. PMID:20643056

A new strategy for the manufacture of a turn-on fluorescent molecularly imprinted polymer (CdTe/SiO2/MIP) receptor for detecting tyrosine phosphopeptide (pTyr peptide) was proposed. The receptor was prepared by the surface imprinting procedure and the epitope approach with silica-capped CdTe quantum dots (QDs) as core substrate and fluorescent signal, phenylphosphonic acid (PPA) as the dummy template, 1-[3-(trimethoxysilyl) propyl] urea as the functional monomer, and octyltrimethoxysilane as the cross-linker. The synthetic CdTe/SiO2/MIP was able to selectively capture the template PPA and corresponding target pTyr peptide with fluorescence enhancement via the special interaction between them and the recognition cavities. The receptor exhibited the linear fluorescence enhancement to pTyr peptide in the range of 0.5-35μM, and the detection limit was 0.37μM. The precision for five replicate detections of pTyr peptide at 20μM was 2.60% (relative standard deviation). Combining the fluorescence property of the CdTe QDs with the merits of the surface imprinting technique and the epitope approach, the receptor not only owned high recognition site accessibility and good binding affinities for target pTyr peptide, but also improved the fluorescence selectivity of the CdTe QDs, as well revealed the feasibility of fabrication of a turn-on fluorescence probe using the surface imprinting procedure and the epitope approach.

A study is presented which shows that /sup 125/I-EGF that was iodinated by lactoperoxidase treatment bound to cells but did not become linked to EGF receptors. /sup 125/I-EGF that was iodinated by chloramine-T treatment or /sup 125/I-EGF that was iodinated by lactoperoxidase treatment and then exposed to chloramine-T, formed linked /sup 125/I-EGF-receptor complexes. Chloramine-T-treated /sup 125/I-EGF remained able to couple to EGF receptors many hours after chloramine-T was removed. These results indicate that chloramine-T ''activates'' /sup 125/I-EGF to a new stable form that couples specifically to EGF receptors. This activation did not occur when Tris middle dot Cl was present during the chloramine-T incubation. Because Tris middle dot Cl is nucleophilic and could moderate the oxidizing effects of chloramine-T, this finding suggests that chloramine-T activates EGF as a result of its ability to oxidize certain amino acid residues in proteins. The specific linkage of chloramine-T-treated /sup 125/I-EGF to EGF receptors provides a convenient, effective method for radiolabeling EGF receptors. The percentage of human fibroblast EGF receptors cross-linked by /sup 125/I-EGF increased to 60% when turnover of EGF receptors at the cell surface was blocked by inhibiting endocytosis with phenylarsine oxide. The linkage of /sup 125/I-thrombin to protease-nexin, a cell-released factor that mediates the binding of /sup 125/I-thrombin to human fibroblasts, is not a chloramine-T artifact. (JMT)

The homophilic cell surfacereceptors CD150 (Slamf1) and CD352 (Slamf6) are known to modulate adaptive immune responses. Although the Th17 response was enhanced in Slamf6(-/-) C57BL/6 mice upon oral infection with Citrobacter rodentium, the pathologic consequences are indistinguishable from an infection of wild-type C57BL/6 mice. Using a reporter-based binding assay, we show that Slamf6 can engage structures on the outer cell membrane of several Gram(-) bacteria. Therefore, we examined whether Slamf6, like Slamf1, is also involved in innate responses to bacteria and regulates peripheral inflammation by assessing the outcome of C. rodentium infections in Rag(-/-) mice. Surprisingly, the pathology and immune responses in the lamina propria of C. rodentium-infected Slamf6(-/-) Rag(-/-) mice were markedly reduced as compared with those of Rag(-/-) mice. Infiltration of inflammatory phagocytes into the lamina propria was consistently lower in Slamf6(-/-) Rag(-/-) mice than in Rag(-/-) animals. Concomitant with the reduced systemic translocation of the bacteria was an enhanced production of IL-22, suggesting that Slamf6 suppresses a mucosal protective program. Furthermore, administering a mAb (330) that inhibits bacterial interactions with Slamf6 to Rag(-/-) mice ameliorated the infection compared with a control antibody. We conclude that Slamf6-mediated interactions of colonic innate immune cells with specific Gram(-) bacteria reduce mucosal protection and enhance inflammation, contributing to lethal colitis that is caused by C. rodentium infections in Rag(-/-) mice.

Cell-cell recognition is a fundamental process that allows cells to coordinate multicellular behaviors. Some microbes, such as myxobacteria, build multicellular fruiting bodies from free-living cells. However, how bacterial cells recognize each other by contact is poorly understood. Here we show that myxobacteria engage in recognition through interactions between TraA cell surfacereceptors, which leads to the fusion and exchange of outer membrane (OM) components. OM exchange is shown to be selective among 17 environmental isolates, as exchange partners parsed into five major recognition groups. TraA is the determinant of molecular specificity because: (i) exchange partners correlated with sequence conservation within its polymorphic PA14-like domain and (ii) traA allele replacements predictably changed partner specificity. Swapping traA alleles also reprogrammed social interactions among strains, including the regulation of motility and conferred immunity from inter-strain killing. We suggest that TraA helps guide the transition of single cells into a coherent bacterial community, by a proposed mechanism that is analogous to mitochondrial fusion and fission cycling that mixes contents to establish a homogenous population. In evolutionary terms, traA functions as a rare greenbeard gene that recognizes others that bear the same allele to confer beneficial treatment. PMID:24244178

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surfacereceptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin β6. The 36 amino acid residues of DSP are sufficient to bind to integrin β6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP (aa183-219) stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin β6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP (aa183-219) in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin β6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop.

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surfacereceptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin β6. The 36 amino acid residues of DSP are sufficient to bind to integrin β6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP aa183-219 stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin β6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP aa183-219 in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin β6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop. PMID:27430624

Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12−/− Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12−/− cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades. PMID:26811477

Six nanosized titanium dioxide powders synthesized from a sulfate process were investigated. The targeted end-use of this powder was for a de-NOx catalyst honeycomb monolith. Alteration of synthesis parameters had resulted principally in differences in soluble ion level and specific surface area of the powders. The goal of this investigation was to understand the role of synthesis parameters in the aggregation behavior of these powders. Investigation via scanning electron microscopy of the powders revealed three different aggregation iterations at specific length scales. Secondary and higher order aggregate strength was investigated via oscillatory stress rheometry as a means of simulating shear conditions encountered during extrusion. G' and G'' were measured as a function of the applied oscillatory stress. Oscillatory rheometry indicated a strong variation as a function of the sulfate level of the particles in the viscoelastic yield strengths. Powder yield stresses ranged from 3.0 Pa to 24.0 Pa of oscillatory stress. Compaction curves to 750 MPa found strong similarities in extrapolated yield point of stage I and II compaction for each of the powders (at approximately 500 MPa) suggesting that the variation in sulfate was greatest above the primary aggregate level. Scanning electron microscopy of samples at different states of shear in oscillatory rheometry confirmed the variation in the linear elastic region and the viscous flow regime. A technique of this investigation was to approach aggregation via a novel perspective: aggregates are distinguished as being loose open structures that are highly disordered and stochastic in nature. The methodology used was to investigate the shear stresses required to rupture the various aggregation stages encountered and investigate the attempt to realign the now free-flowing constituents comprising the aggregate into a denser configuration. Mercury porosimetry was utilized to measure the pore size of the compact resulting from

Background Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum), named Simplagrin. Methods and Findings Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI) with high affinity (2–15 nM), and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα2β1 interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (KD 11 nM) similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation in vivo without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease. Conclusion Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods. PMID:24921659

The ability of the ocular surface to respond to pathogens is in part attributed to toll-like receptors (TLRs) that recognize conserved motifs on various microbes. This study examines TLR expression on various ocular surface cells, if TLR agonists can modulate the expression of antimicrobial peptides (AMPs), human beta defensins (hBD) and cathelicidin (hCAP-18/LL-37) which maybe functionally active against Pseudomonas aeruginosa (PA) and if TLR agonists or AMPs can modulate TLR mRNA expression. TLR1-10 mRNA expression was examined in corneal epithelial, corneal stromal cells and conjunctival epithelial cells by RT-PCR. To confirm protein expression flow cytometry or immunostaining was performed for selected TLRs on some cell cultures. Ocular surface cells were cultured with a range of TLR agonists and then hBD-1, 2, 3, or hCAP-18 mRNA and protein expression was determined by RT-PCR and immunoblotting. In some experiments, cells were cultured with a cocktail of agonists for TLR3, 5 and 6/2 and the antimicrobial activity of the culture media was tested against PA. TLR mRNA expression was also examined in primary human corneal epithelial cells (HCEC) treated with either 3 μg/ml of hBD-2, 5 μg/ml of LL-37 or TLR4, 5 and 9 agonists. Overall, the ocular surface cells expressed mRNA for most of the TLRs but some differences were found. TLR2 was not detected in corneal fibroblasts, TLR4 was not detected in primary cultured or freshly isolated HCEC, TLR5 was not detected in conjunctival epithelial cells (IOBA-NHC) and corneal fibroblasts, TLR7 was not detected in freshly isolated HCEC and TLR10 was not detected in HCEC and IOBA-NHC. TLR8 mRNA was not expressed by any of the samples tested. Immunostaining of cadaver corneas revealed TLR5 and 9 expression throughout the cornea while TLR3 was significantly expressed only in the epithelium. Flow cytometry and immunostaining revealed cultured fibroblasts expressed TLR9 but had no significant TLR3 expression. hBD-2 expression

The superfamily of G-protein-coupled receptors (GPCR) conveys signals in response to various endogenous and exogenous stimuli. Consequently, GPCRs are the most important drug targets. CCR10, the receptor for the chemokines CCL27/CTACK and CCL28/MEC, belongs to the chemokine receptor subfamily of GPCRs and is thought to function in immune responses and tumour progression. However, there is only limited information on the intracellular regulation of CCR10. We find that S100A10, a member of the S100 family of Ca2+ binding proteins, binds directly to the C-terminal cytoplasmic tail of CCR10 and that this interaction regulates the CCR10 cell surface presentation. This identifies S100A10 as a novel interaction partner and regulator of CCR10 that might serve as a target for therapeutic intervention. PMID:26941067

Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0-75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5 kg mol−1 methoxy-terminated PEG corona. While a 5 kg mol−1 methoxy-terminated PEG corona prevents non-specific uptake, a 1.8 kg mol−1 methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associate with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This

Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0-75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5kgmol(-1) methoxy-terminated PEG corona. While a 5kgmol(-1) methoxy-terminated PEG corona prevents non-specific uptake, a 1.8kgmol(-1) methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associated with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This

alpha 2-Adrenergic receptors are present on human erythroleukemia (HEL) cells, both on the cell surface and in a sequestered compartment. In the current study we show that benextramine, a hydrophilic irreversible antagonist, can be used to investigate alpha 2-adrenergic receptor compartmentation in these cells. In membranes prepared from HEL cells, benextramine competed for all alpha 2-adrenergic receptors ( (/sup 3/H)yohimbine sites). In intact cells, at 4 degrees, benextramine exhibited a biphasic competition curve for alpha 2-adrenergic receptors, with EC50 values of approximately 10 microM and greater than 1 mM for the high and low affinity components, respectively. We propose that the alpha 2-adrenergic receptors preferentially blocked by benextramine are those on the surface of the cell, whereas those with low affinity are sequestered receptors because: 1) only epinephrine-accessible sites are removed by prior treatment of cells with benextramine, 2) a preparation enriched with surface membranes is also enriched in receptors with a high affinity for benextramine; and 3) after blockade of cell surfacereceptors (54 +/- 6% of total sites, n = 7) by benextramine, the ability of the alpha 2-adrenergic agonists epinephrine and UK-14,304 to inhibit forskolin-stimulated cAMP accumulation is lost. The latter result implies that only cell surface and not sequestered receptors are functionally coupled to adenylate cyclase. The return of receptors from the sequestered compartment to the cell surface and the recovery of alpha 2-adrenergic receptor function were measured after HEL cells were treated with benextramine (50 microM for 1 hr at 4 degrees). The recovery of receptor binding (t1/2 = 25 min) was somewhat slower than the recovery of function (t1/2 approximately 8 min).

Background The C1q domain containing (C1qDC) proteins refer to a family of all proteins that contain the globular C1q (gC1q) domain, and participate in a series of immune responses depending on their gC1q domains to bind a variety of self and non-self binding ligands. Methodology In the present study, the mRNA expression patterns, localization, and activities of a C1qDC protein from scallop Chlamys farreri (CfC1qDC) were investigated to understand its possible functions in innate immunity. The relative expression levels of CfC1qDC mRNA in hemocytes were all significantly up-regulated after four typical PAMPs (LPS, PGN, β-glucan and polyI:C) stimulation. During the embryonic development of scallop, the mRNA transcripts of CfC1qDC were detected in all the stages, and the expression level was up-regulated from D-hinged larva and reached the highest at eye-spot larva. The endogenous CfC1qDC was dominantly located in the hepatopancreas, gill, kidney and gonad of adult scallop through immunofluorescence. The recombinant protein of CfC1qDC (rCfC1qDC) could not only bind various PAMPs, such as LPS, PGN, β-glucan as well as polyI:C, but also enhance the phagocytic activity of scallop hemocytes towards Escherichia coli. Meanwhile, rCfC1qDC could interact with human heat-aggregated IgG, and this interaction could be inhibited by LPS. Conclusions All these results indicated that CfC1qDC in C. farreri not only served as a PRR involved in the PAMPs recognition, but also an opsonin participating in the clearance of invaders in innate immunity. Moreover, the ability of CfC1qDC to interact with immunoglobulins provided a clue to understand the evolution of classical pathway in complement system. PMID:22905248

Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies.

Human dopamine D2long and D3 receptors were modified by N-terminal addition of SNAP or CLIP forms of O6-alkylguanine-DNA-alkyltransferase plus a peptide epitope tag. Cells able to express each of these four constructs only upon addition of an antibiotic were established and used to confirm regulated and inducible control of expression, the specificity of SNAP and CLIP tag covalent labeling reagents, and based on homogenous time-resolved fluorescence resonance energy transfer, the presence of cell surface D2long and D3 receptor homomers. Following constitutive expression of reciprocal constructs, potentially capable of forming and reporting the presence of cell surface D2long-D3 heteromers, individual clones were assessed for levels of expression of the constitutively expressed protomer. This was unaffected by induction of the partner protomer and the level of expression of the partner required to generate detectable cell surface D2long–D3 heteromers was defined. Such homomers and heteromers were found to co-exist and using a reconstitution of function approach both homomers and heteromers of D2long and D3 receptors were shown to be functional, potentially via trans-activation of associated G protein. These studies demonstrate the ability of dopamine D2long and D3 receptors to form both homomers and heteromers, and show that in cells expressing each subtype a complex mixture of homomers and heteromers co-exists at steady state. These data are of potential importance both to disorders in which D2long and D3 receptors are implicated, like schizophrenia and Parkinson disease, and also to drugs exerting their actions via these sites. PMID:22291025

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential. PMID:26629902

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.

The formation of protein aggregates is a feature of several diseases and is a problem during the manufacture of biopharmaceutical and protein based food products. During processing, stability may become compromised leading to the condensation of proteins to form non-native aggregates. The aim of this work is to induce aggregation on model proteins by the imposition of a particular stress to evaluate the extent of aggregation and to assess the degree of structural change to the protein. Aggregation of two proteins, lysozyme and bovine serum albumin has been induced by several mechanisms. Using various techniques (electrophoresis, HPLC, spectroscopic analysis, and microscopic techniques) both the level of aggregation extent of protein unfolding has been investigated for a range of solution conditions. Our results show that the amount of aggregation depends strongly on the mechanism by which non-native aggregation proceeds, and within each mechanism, solution conditions are an important factor. With the exception of aggregation by self-association (which is concentration dependent), the appearance of aggregation is driven by structural changes induced by the applied stress (heat, chemical denaturant, oxidation or contact with a surface). Author would like to acknowledge support from Science Foundation Ireland (SFI), National University of Maynooth John and Pat Hume Scholarship.

The epidermal growth factor receptor is involved in morphogenesis, proliferation and cell migration. Its up-regulation during tumorigenesis makes this receptor an interesting therapeutic target. In the absence of the ligand, the inhibition of phosphatidic acid phosphohydrolase activity by propranolol treatment leads to internalization of empty/inactive receptors. The molecular events involved in this endocytosis remain unknown. Here, we quantified the effects of propranolol on the mobility of single quantum-dot labelled receptors before the actual internalization took place. The single receptors showed a clear stop-and-go motion; their diffusive tracks were continuously interrupted by sub-second stalling events, presumably caused by transient clustering. In the presence of propranolol we found that: i) the diffusion rate reduced by 22 %, which indicates an increase in drag of the receptor. Atomic force microscopy measurements did not show an increase of the effective membrane tension, such that clustering of the receptor remains the likely mechanism for its reduced mobility. ii) The receptor got frequently stalled for longer periods of multiple seconds, which may signal the first step of the internalization process.

The epidermal growth factor receptor is involved in morphogenesis, proliferation and cell migration. Its up-regulation during tumorigenesis makes this receptor an interesting therapeutic target. In the absence of the ligand, the inhibition of phosphatidic acid phosphohydrolase activity by propranolol treatment leads to internalization of empty/inactive receptors. The molecular events involved in this endocytosis remain unknown. Here, we quantified the effects of propranolol on the mobility of single quantum-dot labelled receptors before the actual internalization took place. The single receptors showed a clear stop-and-go motion; their diffusive tracks were continuously interrupted by sub-second stalling events, presumably caused by transient clustering. In the presence of propranolol we found that: i) the diffusion rate reduced by 22 %, which indicates an increase in drag of the receptor. Atomic force microscopy measurements did not show an increase of the effective membrane tension, such that clustering of the receptor remains the likely mechanism for its reduced mobility. ii) The receptor got frequently stalled for longer periods of multiple seconds, which may signal the first step of the internalization process. PMID:24349439

The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewis(x) trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewis(x) and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewis(x) is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis.

Combinatorial library screens can identify a suitable ligand for a biological target of interest out of thousands or even millions of compounds, and can play a key role in the modern drug development process. While conventional high-throughput cell screens based on functional assays require expensive robotics, simple on-bead combinatorial assays for ligand binding to the target protein can be done far more cheaply. This article describes one such assay, developed using combinatorial peptoid libraries for targeting integral membrane receptors or other cell surface-exposed molecules. In addition to the reduced cost, a unique advantage of this assay is the direct identification of the most selective ligands for a cell surfacereceptor that is expressed in its natural environment. PMID:22582145

Many false positives in early drug discovery owe to nonspecific inhibition by colloid-like aggregates of organic molecules. Despite their prevalence, little is known about aggregate concentration, structure, or dynamic equilibrium; the binding mechanism, stoichiometry with, and affinity for enzymes remain uncertain. To investigate the elementary question of concentration, we counted aggregate particles using flow cytometry. For seven aggregate-forming molecules, aggregates were not observed until the concentration of monomer crossed a threshold, indicating a “critical aggregation concentration” (CAC). Above the CAC, aggregate count increased linearly with added organic material, while the particles dispersed when diluted below the CAC. The concentration of monomeric organic molecule is constant above the CAC, as is the size of the aggregate particles. For two compounds that form large aggregates, nicardipine and miconazole, we measured particle numbers directly by flow cytometry, determining that the aggregate concentration just above the CAC ranged from 5 to 30 fM. By correlating inhibition of an enzyme with aggregate count for these two drugs, we determined that the stoichiometry of binding is about 10 000 enzyme molecules per aggregate particle. Using measured volumes for nicardipine and miconazole aggregate particles (2.1 × 1011 and 4.7 × 1010 Å3, respectively), computed monomer volumes, and the observation that past the CAC all additional monomer forms aggregate particles, we find that aggregates are densely packed particles. Finally, given their size and enzyme stoichiometry, all sequestered enzyme can be comfortably accommodated on the surface of the aggregate. PMID:18588298

This is a follow up on the preceding presentation by Crosson. The grid size for remote microwave measurements is much coarser than the hydrological model computational grids. To validate the hydrological models with measurements we propose mechanisms to aggregate the hydrological model outputs for soil moisture to allow comparison with measurements. Weighted neighborhood averaging methods are proposed to facilitate the comparison. We will also discuss such complications as misalignment, rotation and other distortions introduced by a generalized sensor image.

Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

Using soluble {sup 123}I-labeled aggregates of human IgG ({sup 123}I-AHIgG) as a probe, we examined the function of the mononuclear phagocyte system in 22 patients with systemic lupus erythematosus (SLE) and 12 healthy controls. In SLE patients, a decreased number of erythrocyte complement receptor type 1 was associated with less binding of {sup 123}I-AHIgG to erythrocytes and a faster initial rate of elimination of {sup 123}I-AHIgG (mean +/- SEM half-maximal clearance time 5.23 +/- 0.2 minutes, versus 6.58 +/- 0.2 minutes in the controls), with possible spillover of the material outside the mononuclear phagocyte system of the liver and spleen. However, multiple regression analysis showed that serum concentrations of IgG were the most important factor predicting the rate of {sup 123}I-AHIgG elimination. IgG concentration may thus reflect immune complex clearance, which in turn, would influence the inflammatory reaction, in SLE.

Amylin is a peptide that aggregates into species that are toxic to pancreatic beta cells, leading to type II diabetes. This study has for the first time quantified amylin association and dissociation kinetics (association constant (ka) = 28.7 ± 5.1 L mol−1 s−1 and dissociation constant (kd) = 2.8 ± 0.6 ×10−4 s−1) using surface plasmon resonance (SPR). Thus far, techniques used for the sizing of amylin aggregates do not cater for the real-time monitoring of unconstrained amylin in solution. In this regard we evaluated recently innovated nanoparticle tracking analysis (NTA). In addition, both SPR and NTA were used to study the effect of previously synthesized amylin derivatives on amylin aggregation and to evaluate their potential as a cell-free system for screening potential inhibitors of amylin-mediated cytotoxicity. Results obtained from NTA highlighted a predominance of 100–300 nm amylin aggregates and correlation to previously published cytotoxicity results suggests the toxic species of amylin to be 200–300 nm in size. The results seem to indicate that NTA has potential as a new technique to monitor the aggregation potential of amyloid peptides in solution and also to screen potential inhibitors of amylin-mediated cytotoxicity. PMID:26019498

The prevalence of nanoparticles in the environment is expected to grow in the coming years due to their increasing pervasiveness in consumer and industrial applications. Once released into the environment, nanoparticles encounter conditions of pH, salinity, UV light, and other solution conditions that may alter their surface characteristics and lead to aggregation. The unique properties that make nanoparticles desirable are a direct consequence of their size and increased surface area. Therefore, it is critical to recognize how aggregation alters the reactive properties of nanomaterials, if we wish to understand how these properties are going to behave once released into the environment. The size and structure of nanoparticle aggregates depend on surrounding conditions, including hydrodynamic ones. Depending on these conditions, aggregates can be large or small, tightly packed or loosely bound. Characterizing and measuring these changes to aggregate morphology is important to understanding the impact of aggregation on nanoparticle reactive properties. Examples of decreased reactivity due to aggregation include the case where tightly packed aggregates have fewer available surface sites compared to loosely packed ones; also, photocatalytic particles embedded in the center of large aggregates will experience less light when compared to particles embedded in small aggregates. However, aggregation also results in an increase in solid-solid interfaces between nanoparticles. This can result in increased energy transfer between neighboring particles, surface passivation, and altered surface tension. These phenomena can lead to an increase in reactivity. The goal of this thesis is to examine the impacts of aggregation on the reactivity of a select group of nanomaterials. Additionally, we examined how aggregation impacts the removal efficiency of fullerene nanoparticles using membrane filtration. The materials we selected to study include ZnS---a metal chalcogenide

Thyroid hormone receptors (TRs) bind as homodimers or heterodimers with retinoid X receptors (RXRs) to DNA elements with diverse orientations of AGGTCA half-sites. We performed a comprehensive x-ray crystal structure-guided mutation analysis of the TR ligand binding domain (TR LBD) surface to map the functional interface for TR homodimers and heterodimers with RXR in the absence and/or in the presence of DNA. We also identified the molecular contacts in TR LBDs crystallized as dimers. The results show that crystal dimer contacts differ from those found in the functional studies. We found that identical TR LBD residues found in helices 10 and 11 are involved in TR homodimerization and heterodimerization with RXR. Moreover, the same TR LBD surface is operative for dimerization with direct repeats spaced by 4 base pairs (DR-4) and with the inverted palindrome spaced by 6 base pairs (F2), but not with TREpal (unspaced palindrome), where homodimers appear to be simply two monomers binding independently to DNA. We also demonstrate that interactions between the TR and RXR DNA binding domains stabilize TR-RXR heterodimers on DR-4. The dimer interface can be functional in the cell, because disruption of key residues impairs transcriptional activity of TRs mediated through association with RXR LBD linked to GAL4 DNA-binding domain.

This book cover the reviews on biomembrane dynamics; recent spectroscopic studies. Topics covered are freeze fracture: Seeing and thinking biological membranes, membrane proteins and receptors: structure and organisation; techniques to determine the transbilayer distribution and mobility of phospholipids in biological membranes, transbilayer organisation of phospholipids in the plasma membranes of pro-erythroblasts and normal and abnormal red cells, aminophospholipid translocation in the erythroctye membrane is mediated by a specific AIP-dependent enzyme; membrane protein interactions, lipid-protein interactions: selectively and receptor binding, membrane fluidity in the regulation of membrane-linked enzymes, the lipid regulation of receptor functions, microheterogencity of biological membrane: structural and functional implications, fusion-fission reactions in biological membranes and in phospholpid bilayers, methods for studying the structure and function of the mitochondrial uncoupling protein, methods for studying metabolite transport in mitochondria, transport of metabolites in mitochondria, membrane gangliosides and allied glycosphingolipids: Biochemical features and physicochemical properties, the use of merocyanine 540 for monitoring aggregation properties of sialogangliosides in solution, hormone reception at the cell surface - an overview, double role for GIP in the stimulus secretion sequence of mast cells and neurophils, tumor promoters and hormone receptor coupling mechanisms in the anterior pituitary. The regulation of hormone-dependent adenylate cyclase in native membranes and systems reconstituted from purified components.- Immunological tools for the study of plasma membrane receptors.

Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulin's two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four “site 1” and six “site 2” analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. The results show that Val43, Phe28, and Val14 (equivalent to site 1) are critical to IGF-1R and IR binding, whereas mutation to alanine of Gln18 affects only IGF-1R and not IR binding. Alanine substitutions at Glu12, Asp15, Phe19, Leu53, and Glu57 analogues resulted in significant (>2-fold) decreases in affinity for both the IGF-1R and IR. Furthermore, taking a novel approach using a monomeric, single-chain minimized IGF-1R we have defined a distinct second binding surface formed by Glu12, Phe19, Leu53, and Glu57 that potentially engages the IGF-1R at one or more of the FnIII domains. PMID:19139090

The rapid rise in natural gas extraction using hydraulic fracturing increases the potential for contamination of surface and ground water from chemicals used throughout the process. Hundreds of products containing more than 750 chemicals and components are potentially used throughout the extraction process, including more than 100 known or suspected endocrine-disrupting chemicals. We hypothesized thataselected subset of chemicalsusedin natural gas drilling operationsandalso surface and ground water samples collected in a drilling-dense region of Garfield County, Colorado, would exhibit estrogen and androgen receptor activities. Water samples were collected, solid-phase extracted, and measured for estrogen and androgen receptor activities using reporter gene assays in human cell lines. Of the 39 unique water samples, 89%, 41%, 12%, and 46% exhibited estrogenic, antiestrogenic, androgenic, and antiandrogenic activities, respectively. Testing of a subset of natural gas drilling chemicals revealed novel antiestrogenic, novel antiandrogenic, and limited estrogenic activities. The Colorado River, the drainage basin for this region, exhibited moderate levels of estrogenic, antiestrogenic, and antiandrogenic activities, suggesting that higher localized activity at sites with known natural gas–related spills surrounding the river might be contributing to the multiple receptor activities observed in this water source. The majority of water samples collected from sites in a drilling-dense region of Colorado exhibited more estrogenic, antiestrogenic, or antiandrogenic activities than reference sites with limited nearby drilling operations. Our data suggest that natural gas drilling operationsmayresult in elevated endocrine-disrupting chemical activity in surface and ground water.

The rapid rise in natural gas extraction using hydraulic fracturing increases the potential for contamination of surface and ground water from chemicals used throughout the process. Hundreds of products containing more than 750 chemicals and components are potentially used throughout the extraction process, including more than 100 known or suspected endocrine-disrupting chemicals. We hypothesized that a selected subset of chemicals used in natural gas drilling operations and also surface and ground water samples collected in a drilling-dense region of Garfield County, Colorado, would exhibit estrogen and androgen receptor activities. Water samples were collected, solid-phase extracted, and measured for estrogen and androgen receptor activities using reporter gene assays in human cell lines. Of the 39 unique water samples, 89%, 41%, 12%, and 46% exhibited estrogenic, antiestrogenic, androgenic, and antiandrogenic activities, respectively. Testing of a subset of natural gas drilling chemicals revealed novel antiestrogenic, novel antiandrogenic, and limited estrogenic activities. The Colorado River, the drainage basin for this region, exhibited moderate levels of estrogenic, antiestrogenic, and antiandrogenic activities, suggesting that higher localized activity at sites with known natural gas-related spills surrounding the river might be contributing to the multiple receptor activities observed in this water source. The majority of water samples collected from sites in a drilling-dense region of Colorado exhibited more estrogenic, antiestrogenic, or antiandrogenic activities than reference sites with limited nearby drilling operations. Our data suggest that natural gas drilling operations may result in elevated endocrine-disrupting chemical activity in surface and ground water.

In the Biacore biosensor, a widely used tool for studying the kinetics of ligand/receptor binding, receptors are commonly localized to the sensor surface through attachment to polymers that extend from the surface to form a layer. The importance of the polymeric layer in analyzing data is controversial. The question of the effect of a binding layer also arises in the case of ligands interacting with binding sites distributed in the extracellular matrix of cells. To identify and quantify the effects of a binding layer on the estimation of association and dissociation rate constants, we derived effective rate coefficients. The expressions show that rate constants determined under the standard assumption that binding takes place on a two-dimensional surface underestimate the true reaction rate constants by a factor that depends on the ratio of the height of the layer to the mean free path of the ligand within the layer. We show that, for typical biological ligands, receptors, cells, and Biacore conditions, the binding layer will affect the interpretation of data only if transport of the ligand in the layer is slowed substantially--by one or two orders of magnitude--relative to transport outside the layer. From existing experiments and theory, it is not clear which Biacore experiments, if any, have transport within the dextran layer reduced to such an extent. We propose a method, based on the effective rate coefficients we have derived, for the experimental determination of ligand diffusion coefficients in a polymeric matrix. PMID:11916835

It is generally known that there are compounds present in the aquatic environment that can disturb endocrine processes, for example via interaction with the endogenous hormone receptors. Most research so far has focused on compounds that bind to the estrogen and/or androgen receptor, but ligands for other hormone receptors might also be present. In this study, a newly completed panel of human cell derived CALUX reporter gene bioassays was utilized to test water extracts for estrogen (ER), as well as androgen (AR), progesterone (PR), and glucocorticoid (GR) receptor mediated transactivation activity. Effluents from industry, hospital, and municipal sewage treatment plants, as well as tap water and different sources of surface water were tested. The CALUX reporter gene panel showed high sensitivity and specificity to known agonists, enabling discrimination between different receptor based endocrine responses present in the aquatic environment. Our results clearly showed the presence of agonistic activity on the ER, as well as on the AR, PR, and GR in the raw and wastewater and surface water extracts. However, no hormone receptor-mediated transactivation was detected in the drinking water or in the blank water. The levels of estrogenic activity were 0.2-0.5 ng E2-equiv/L for surface water and 0.4-1.0 ng E2-equiv/L for municipal effluents, which was consistent with previous studies. Surprisingly, the other hormonal activities were found to be present in similar or much higher levels. Most notably, glucocorticoid-like activity was detected in all samples, at surprisingly high levels ranging from 0.39-1.3 ng Dex-equiv/L in surface water and 11-243 ng Dex-equiv/L in effluents. When regarding the fact that dexamethasone in the GR CALUX bioassay is a factor 12 more potent than the natural hormone cortisol, results expressed as cortisol equivalents would range up to 2900 ng cortisol equiv/L. Further studies are needed to establish the identity of the active compounds and to

Dendritic cells (DCs) are highly potent stimulators of the immune system, and their contribution as such to the pathogenesis of corneal and ocular surface inflammatory disease has been well established. These vigorous antigen-presenting cells are reliant upon their effective migration from peripheral tissues (e.g., those of the ocular surface) to the lymphoid organs, where immune responses are triggered and can then cause disease. The chemokine receptor CCR7 expressed on DCs has emerged as the master mediator of this highly complex migratory process, and thus it is important in causing corneal and ocular surface inflammation. Furthermore, CCR7 has received considerable attention as a potential therapeutic target, as topically instilled antagonists of this receptor are quite effective therapeutically in a mouse model of ocular allergy. These findings and more are reviewed in the current article. In addition, the understanding regarding CCR7 function in mice and humans, and the biology of DCs that populate the ocular surface are also detailed herein. The involvement of DCs and their expression of CCR7 in corneal and ocular surface diseases such as in ocular allergy, dry eye disease, immune rejection and more, are also reviewed here.

An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin.

Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3−/− and Mgat5−/− mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2. PMID:21715322

Monoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF-R) as well as to a soluble, truncated form of the receptor (NGF-Rt). The antibodies were generated against partially purified NGF-Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk-) that expresses large numbers of the low affinity form of the human NGF-R on its cell surface. Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surfacereceptor covalently cross-linked to {sup 125}I-NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl,k subclass. All monoclonal antibodies bound to both NGF-R and NGF-Rt. Two monoclonal antibodies (VIID1, XIF1) immunoblotted the NGF-R from E9b cell preparations resolved on non-reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. The antibodies immunoprecipitated NGF-R from both E9b cells and from SH-SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF-Rt, but did not cross-react with NGF-R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF-Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2-site RISA. The 2-site RISA can be used to rapidly quantitate NGF-R and NGF-Rt in large numbers of biological samples in the absence of added {sup 125}I-labeled NGF.

Plants must adapt to their environment and require mechanisms for sensing their surroundings and responding appropriately. An expanded family of more than 200 leucine-rich repeat (LRR) receptor kinases (LRR-RKs) transduces fluctuating and often contradictory signals from the environment into changes in nuclear gene expression. Two LRR-RKs, BRASSINOSTEROID INSENSITIVE 1 (BRI1), a steroid receptor, and FLAGELLIN SENSITIVE 2 (FLS2), an innate immune receptor that recognizes bacterial flagellin, act cooperatively to partition necessary growth-defense trade-offs. BRI1 and FLS2 share common signaling components and slightly different activation mechanisms. BRI1 and FLS2 are paradigms for understanding the signaling mechanisms of LRR-containing receptors in plants.

Urokinase plasminogen activator receptor (u-PAR) binds urokinase plasminogen activator (u-PA) and participates in plasminogen activation in addition to modulating several cellular processes such as adhesion, proliferation, and migration. u-PAR is susceptible to proteolysis by its cognate ligand and several other proteases. To elucidate the biological significance of receptor cleavage by u-PA, we engineered and expressed a two-chain urokinase plasminogen activator (tcu-PA) cleavage-resistant u-PAR (cr-u-PAR). This mutated receptor was similar to wild-type u-PAR in binding u-PA and initiating plasminogen activation. However, cr-u-PAR exhibited accelerated internalization and resurfacing due to direct association with the endocytic receptor alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein in the absence of the enzyme x inhibitor complex of tcu-PA and plasminogen activator inhibitor-1 (tcu-PA.PAI-1). cr-u-PAR-expressing cells had enhanced migration compared with wild-type u-PAR-expressing cells, and cr-u-PAR was less sensitive to chymotrypsin cleavage as compared with wt u-PAR. Our studies suggest that these mutations in the linker region result in a rearrangement within the cr-u-PAR structure that makes it resemble its ligand-bound form. This constitutively active variant may mimic highly glycosylated cleavage-resistant u-PAR expressed in certain highly malignant cancer-cells.

Following activation by their cognate ligands, cytokine receptors undergo intracellular routing towards lysosomes where they are degraded. Cytokine receptor signaling does not terminate at the plasma membrane, but continues throughout the endocytotic pathway. The modes of internalization and intracellular trafficking of specific receptors, the level of recycling towards the plasma membrane, the type of protein modifications (phosphorylation, ubiquitination) and the enzymes involved in these processes are remarkably diverse. This heterogeneity may contribute to the fine-tuning of signal amplitudes and duration from different receptors. The colony-stimulating factor 3 receptor (CSF3R) is unique for its balanced signaling output, first leading to proliferation of myeloid progenitors, followed by a cell cycle arrest and granulocytic differentiation. The mechanisms associated with CSF3R signal modulation, involving receptor lysine ubiquitination and redox-controlled phosphatase activities, are to a large extent confined to the signaling endosome. Interactions between signaling endosomes and the endoplasmic reticulum play a key role in this process. Here, we review the mechanisms of intracellular routing of CSF3R, their consequences for myeloid blood cell development and their implications for myeloid diseases.

We introduce and define the concept of mean aggregation of a collection of n numbers. We point out that the lack of associativity of this operation compounds the problem of the extending mean of n numbers to n+1 numbers. The closely related concepts of self identity and the centering property are introduced as one imperative for extending mean aggregation operators. The problem of weighted mean aggregation is studied. A new concept of prioritized mean aggregation is then introduced. We next show that the technique of selecting an element based upon the performance of a random experiment can be considered as a mean aggregation operation.

We have simulated the structure of kinetically irreversible protein aggregates in two-dimensional space using a lattice-based Monte-Carlo routine. Our model specifically accounts for the intermolecular interactions between hydrophobic and hydrophilic protein surfaces and a polar solvent. The simulations provide information about the aggregate density, the types of inter-monomer contacts and solvent content within the aggregates, the type and extent of solvent exposed perimeter, and the short- and long-range order all as a function of (i) the extent of monomer hydrophobic surface area and its distribution on the model protein surface and (ii) the magnitude of the hydrophobic-hydrophobic contact energy. An increase in the extent of monomer hydrophobic surface area resulted in increased aggregate densities with concomitant decreased system free energies. These effects are accompanied by increases in the number of hydrophobic-hydrophobic contacts and decreases in the solvent-exposed hydrophobic surface area of the aggregates. Grouping monomer hydrophobic surfaces in a single contiguous stretch resulted in lower aggregate densities and lower short range order. More favorable hydrophobic-hydrophobic contact energies produced structures with higher densities but the number of unfavorable protein-protein contacts was also observed to increase; greater configurational entropy produced the opposite effect. Properties predicted by our model are in good qualitative agreement with available experimental observations. Images FIGURE 6 FIGURE 13 PMID:8061184

Flash NanoPrecipitation (FNP) is a novel approach for producing functional nanoparticles stabilized by amphiphilic block copolymers. FNP involves the rapid mixing of a hydrophobic active (organic) and an amphiphilic di-block copolymer with a non-solvent (water) and subsequent co-precipitation of nanoparticles composed of both the organic and copolymer. During this process, the particle size distribution (PSD) is frozen and stabilized by the hydrophilic portion of the amphiphilic di-block copolymer residing on the particle surface. That is, the particle growth is kinetically arrested and thus a narrow PSD can be attained. To model the co-precipitation process, a bivariate population balance equation (PBE) has been formulated to account for the competitive aggregation of the organic and copolymer versus pure organic-organic or copolymer-copolymer aggregation. Aggregation rate kernels have been derived to account for the major aggregation events: free coupling, unimer insertion, and aggregate fusion. The resulting PBE is solved both by direct integration and by using the conditional quadrature method of moments (CQMOM). By solving the competitive aggregation model under well-mixed conditions, it is demonstrated that the PSD is controlled primarily by the copolymer-copolymer aggregation process and that the energy barrier to aggregate fusion plays a key role in determining the PSD. It is also shown that the characteristic aggregation times are smaller than the turbulent mixing time so that the FNP process is always mixing limited.

Estrogen receptor α (ERα) and Specificity protein 1 (Sp1) are transcription factors (TF) that are involved in regulating progesterone receptor (PR) gene expression through cooperative interactions with DNA. The natural composite DNA +571 ERE/Sp1 site in promoter A of the progesterone receptor contains a half-site of estrogen response elements (½ERE) upstream of two Sp1 binding sites (the proximal Sp1 (Sp1/P) and distal Sp1 (Sp1/D)) with a 4 bp spacer. Here, we have developed a protocol for studying the cooperative interaction of Sp1 and ERα with the composite DNA of +571 ERE/Sp1 site using Biacore T200, a high sensitivity surface plasmon resonance spectroscopy. With this protocol, we have concluded that Sp1 binding enhances the overall ERα binding to the composite DNA. We have also determined the optimal spacer distance between the ½ERE and Sp1/D for the best cooperative protein binding. This study is pivotal in guiding the bioinformatics simulation to yield an exact model of the spacer dependency of the transcription factor/cofactor-DNA interactions, which is important for understanding the nuclear receptor regulating activity through other coactivators.

Several somatic mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have been identified that predict clinical response of non-small-cell lung carcinoma (NSCLC) patients to gefitinib. To test the hypothesis that these mutations cause constitutive EGF receptor signaling, and to investigate its mechanistic basis, we expressed representative examples in a null background and analysed their biochemical properties. Each mutation caused significant EGF-independent tyrosine phosphorylation of EGFR, and allowed the receptor to promote Ba/F3 cell mitogenesis in the absence of EGF, arguing that these are oncogenic mutations. Active mutated receptors are present at the cell surface and are fully competent to bind EGF. Recent structural studies show that the inactive EGFR tyrosine kinase domain is autoinhibited by intramolecular interactions between its activation loop and alphaC helix. We find that mutations predicted to disrupt this autoinhibitory interaction (including several that have not been described in NSCLC) elevate EGF-independent tyrosine kinase activity, thus providing new insight into how somatic mutations activate EGFR and other ErbB family members.

Crushed stone and sand and gravel are the two main sources of natural aggregates. These materials are commonly used construction materials and frequently can be interchanged with one another. They are widely used throughout the United States, with every State except two producing crushed stone. Together they amount to about half the mining volume in the United States. Approximately 96 percent of sand and gravel and 77 percent of the crushed stone produced in the United States are used in the construction industry. Natural aggregates are widely distributed throughout the United States in a variety of geologic environments. Sand and gravel deposits commonly are the results of the weathering of bedrock and subsequent transportation and deposition of the material by water or ice (glaciers). As such, they commonly occur as river or stream deposits or in glaciated areas as glaciofluvial and other deposits. Crushed stone aggregates are derived from a wide variety of parent bedrock materials. Limestone and other carbonates account for approximately three quarters of the rocks used for crushed stone, with granite and other igneous rocks making up the bulk of the remainder. Limestone deposits are widespread throughout the Central and Eastern United States and are scattered in the West. Granites are widely distributed in the Eastern and Western United States, with few exposures in the Midwest. Igneous rocks (excluding granites) are largely concentrated in the Western United States and in a few isolated localities in the East. Even though natural aggregates are widely distributed throughout the United States, they are not universally available for consumptive use. Some areas are devoid of sand and gravel, and potential sources of crushed stone may be covered with sufficient unconsolidated material to make surface mining impractical. In some areas many aggregates do not meet the physical property requirements for certain uses, or they may contain mineral constituents that react

Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was 32P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (H11 and 111.6) and one phosphospecific EGF receptor antibody (pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82:1, exceeding the signal-to-background measured on the ELISA plate (<48:1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the transmembrane protein

Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection. PMID:26917727

In contaminant travel from ground surface to groundwater receptors, the time taken in travelling through the unsaturated zone is known as the unsaturated zone time lag. Depending on the situation, this time lag may or may not be significant within the context of the overall problem. A method is presented for assessing the importance of the unsaturated zone in the travel time from source to receptor in terms of estimates of both the absolute and the relative advective times. A choice of different techniques for both unsaturated and saturated travel time estimation is provided. This method may be useful for practitioners to decide whether to incorporate unsaturated processes in conceptual and numerical models and can also be used to roughly estimate the total travel time between points near ground surface and a groundwater receptor. This method was applied to a field site located in a glacial aquifer system in Ontario, Canada. Advective travel times were estimated using techniques with different levels of sophistication. The application of the proposed method indicates that the time lag in the unsaturated zone is significant at this field site and should be taken into account. For this case, sophisticated and simplified techniques lead to similar assessments when the same knowledge of the hydraulic conductivity field is assumed. When there is significant uncertainty regarding the hydraulic conductivity, simplified calculations did not lead to a conclusive decision.

Axon regeneration in the CNS is largely unsuccessful due to excess inhibitory extrinsic factors within lesion sites together with an intrinsic inability of neurons to regrow following injury. Recent work demonstrates that forced expression of certain neuronal transmembrane receptors can recapitulate neuronal growth resulting in successful growth within and through inhibitory lesion environments. More specifically, neuronal expression of integrin receptors such as alpha9beta1 integrin which binds the extracellular matrix glycoprotein tenascin-C, trk receptors such as trkB which binds the neurotrophic factor BDNF, and receptor PTPσ which binds chondroitin sulphate proteoglycans, have all been show to significantly enhance regeneration of injured axons. We discuss how reintroduction of these receptors in damaged neurons facilitates signalling from the internal environment of the cell with the external environment of the lesion milieu, effectively resulting in growth and repair following injury. In summary, we suggest an appropriate balance of intrinsic and extrinsic factors are required to obtain substantial axon regeneration. PMID:28197173

Adsorption of the temperature-responsive polymer hydroxypropylmethylcellulose (HPMC) from an aqueous solution onto hydrophobized silica was followed well above the bulk instability temperature (T(2)) in temperature cycle experiments. Two complementary techniques, QCM-D and ellipsometry, were utilized simultaneously to probe the same substrate immersed in polymer solution. The interfacial processes were correlated with changes in polymer aggregation and viscosity of polymer solutions, as monitored by light scattering and rheological measurements. The simultaneous use of ellipsometry and QCM-D, and the possibility to follow layer properties up to 80 °C, well above the T(2) temperature, are both novel developments. A moderate increase in adsorbed amount with temperature was found below T(2), whereas a significant increase in the adsorbed mass and changes in layer properties were observed around the T(2) temperature where the bulk viscosity increases significantly. Thus, there is a clear correlation between transition temperatures in the adsorbed layer and in bulk solution, and we discuss this in relation to a newly proposed model that considers competition between aggregation and adsorption/deposition. A much larger temperature response above the T(2) temperature was found for adsorbed layers of HPMC than for layers of methyl cellulose. Possible reasons for this are discussed.

IL-15 is a short chain, four-alpha helix cytokine that shares some biological function with IL-2. One striking difference between IL-2 and IL-15 is the ability of monocytes to express IL-15 on their cell surface after activation. In the current study we have investigated the ability of human monocyte cell surface IL-15 to participate in reverse signaling. Cross-linking anti-IL-15 Abs were used as a surrogate ligand for surface IL-15 engagement. Ligation of cell surface-expressed IL-15 induced monocyte adhesion that required the activity of small m.w. GTPases. Reverse signals through surface IL-15 activated the Rho-GTPase Rac3. In addition, engagement of cell surface IL-15 was found to activate a number of signaling pathways, including both extracellular signal-regulated kinase 1/2 and p38, and resulted in the secretion of IL-8. IL-8 production required mitogen-activated protein kinase activity. Thus, the current study has established that cell surface IL-15 is more than just a ligand; it can function as a receptor and participate in reverse signaling that results in cellular adhesion and production of inflammatory cytokines.

Vitamin A and its derivatives (retinoids) play diverse and crucial functions from embryogenesis to adulthood and are used as therapeutic agents in human medicine for eye and skin diseases, infections and cancer. Plasma retinol binding protein (RBP) is the principal and specific vitamin A carrier in the blood and binds vitamin A at 1:1 ratio. STRA6 is the high-affinity membrane receptor for RBP and mediates cellular vitamin A uptake. STRA6 null mice have severely depleted vitamin A reserves for vision and consequently have vision loss, even under vitamin A sufficient conditions. STRA6 null humans have a wide range of severe pathological phenotypes in many organs including the eye, brain, heart and lung. Known membrane transport mechanisms involve transmembrane pores that regulate the transport of the substrate (e.g., the gating of ion channels). STRA6 represents a new type of membrane receptor. How this receptor interacts with its transport substrate vitamin A and the functions of its nine transmembrane domains are still completely unknown. These questions are critical to understanding the molecular basis of STRA6's activities and its regulation. We employ acute chemical modification to introduce chemical side chains to STRA6 in a site-specific manner. We found that modifications with specific chemicals at specific positions in or near the transmembrane domains of this receptor can almost completely suppress its vitamin A transport activity. These experiments provide the first evidence for the existence of a transmembrane pore, analogous to the pore of ion channels, for this new type of cell-surfacereceptor.

We study the link between atomic motion and exciton transport in flexible Rydberg aggregates, assemblies of highly excited light alkali-metal atoms, for which motion due to dipole-dipole interaction becomes relevant. In two one-dimensional atom chains crossing at a right angle adiabatic exciton transport is affected by a conical intersection of excitonic energy surfaces, which induces controllable nonadiabatic effects. A joint exciton-motion pulse that is initially governed by a single energy surface is coherently split into two modes after crossing the intersection. The modes induce strongly different atomic motion, leading to clear signatures of nonadiabatic effects in atomic density profiles. We have shown how this scenario can be exploited as an exciton switch, controlling direction and coherence properties of the joint pulse on the second of the chains [K. Leonhardt et al., Phys. Rev. Lett. 113, 223001 (2014), 10.1103/PhysRevLett.113.223001]. In this article we discuss the underlying complex dynamics in detail, characterize the switch, and derive our isotropic interaction model from a realistic anisotropic one with the addition of a magnetic bias field.

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

Distinct patterns in neuronal firing are observed between classes of cutaneous afferents. Such differences may be attributed to end-organ morphology, distinct ion-channel complements, and skin microstructure, among other factors. Even for just the slowly adapting type I afferent, the skin's mechanics for a particular specimen might impact the afferent's firing properties, especially given the thickness and elasticity of skin can change dramatically over just days. Here, we show computationally that the skin can reliably convey indentation magnitude, rate, and spatial geometry to the locations of tactile receptors even amid changes in skin's structure. Using finite element analysis and neural dynamics models, we considered the skin properties of six mice that span a representative cohort. Modeling the propagation of the surface stimulus to the interior of the skin demonstrated that there can be large variance in stresses and strains near the locations of tactile receptors, which can lead to large variance in static firing rate. However, variance is significantly reduced when the stimulus tip is controlled by surface pressure and compressive stress is measured near the end organs. This particular transformation affords the least variability in predicted firing rates compared with others derived from displacement, force, strain energy density, or compressive strain. Amid changing skin mechanics, stimulus control by surface pressure may be more naturalistic and optimal and underlie how animals actively explore the tactile environment.

A simple, novel and generally applicable method to demonstrate structure-activity associations of a group of biologically interesting compounds in relation to receptor binding is described. This method is useful for undergraduates and graduate students in medicinal chemistry and computer modeling programs.

Layers of dormant endospores of Bacillus subtilis HA101 were applied to eight different spacecraft materials and exposed to martian conditions of low pressure (8.5 mbar), low temperature (-10°C), and high CO2 gas composition and irradiated with a Mars-normal ultraviolet (UV-visible- near-infrared spectrum. Bacterial layers were exposed to either 1 min or 1 h of Mars-normal UV irradiation, which simulated clear-sky conditions on equatorial Mars (0.1 tau). When exposed to 1 min of Mars UV irradiation, the numbers of viable endospores of B. subtilis were reduced three to four orders of magnitude for two brands of aluminum (Al), stainless steel, chemfilm-treated Al, clear-anodized Al, and black-anodized Al coupons. In contrast, bacterial survival was reduced only one to two orders of magnitude for endospores on the non-metal materials astroquartz and graphite composite when bacterial endospores were exposed to 1 min of Mars UV irradiation. When bacterial monolayers were exposed to 1 h of Mars UV irradiation, no viable bacteria were recovered from the six metal coupons listed above. In contrast, bacterial survival was reduced only two to three orders of magnitude for spore layers on astroquartz and graphite composite exposed to 1 h of Mars UV irradiation. Scanning electron microscopy images of the bacterial monolayers on all eight spacecraft materials revealed that endospores of B. subtilis formed large aggregates of multilayered spores on astroquartz and graphite composite, but not on the other six spacecraft materials. It is likely that the formation of multilayered aggregates of endospores on astroquartz and graphite composite is responsible for the enhanced survival of bacterial cells on these materials.

Layers of dormant endospores of Bacillus subtilis HA101 were applied to eight different spacecraft materials and exposed to martian conditions of low pressure (8.5 mbar), low temperature (-10 degrees C), and high CO(2) gas composition and irradiated with a Mars-normal ultraviolet (UV-visible- near-infrared spectrum. Bacterial layers were exposed to either 1 min or 1 h of Mars-normal UV irradiation, which simulated clear-sky conditions on equatorial Mars (0.1 tau). When exposed to 1 min of Mars UV irradiation, the numbers of viable endospores of B. subtilis were reduced three to four orders of magnitude for two brands of aluminum (Al), stainless steel, chemfilm-treated Al, clear-anodized Al, and black-anodized Al coupons. In contrast, bacterial survival was reduced only one to two orders of magnitude for endospores on the non-metal materials astroquartz and graphite composite when bacterial endospores were exposed to 1 min of Mars UV irradiation. When bacterial monolayers were exposed to 1 h of Mars UV irradiation, no viable bacteria were recovered from the six metal coupons listed above. In contrast, bacterial survival was reduced only two to three orders of magnitude for spore layers on astroquartz and graphite composite exposed to 1 h of Mars UV irradiation. Scanning electron microscopy images of the bacterial monolayers on all eight spacecraft materials revealed that endospores of B. subtilis formed large aggregates of multilayered spores on astroquartz and graphite composite, but not on the other six spacecraft materials. It is likely that the formation of multilayered aggregates of endospores on astroquartz and graphite composite is responsible for the enhanced survival of bacterial cells on these materials.

Layers of dormant endospores of Bacillus subtilis HA101 were applied to eight different spacecraft materials and exposed to martian conditions of low pressure (8.5 mbar), low temperature (-10 degrees C), and high CO(2) gas composition and irradiated with a Mars-normal ultraviolet (UV-visible- near-infrared spectrum. Bacterial layers were exposed to either 1 min or 1 h of Mars-normal UV irradiation, which simulated clear-sky conditions on equatorial Mars (0.1 tau). When exposed to 1 min of Mars UV irradiation, the numbers of viable endospores of B. subtilis were reduced three to four orders of magnitude for two brands of aluminum (Al), stainless steel, chemfilm-treated Al, clear-anodized Al, and black-anodized Al coupons. In contrast, bacterial survival was reduced only one to two orders of magnitude for endospores on the non-metal materials astroquartz and graphite composite when bacterial endospores were exposed to 1 min of Mars UV irradiation. When bacterial monolayers were exposed to 1 h of Mars UV irradiation, no viable bacteria were recovered from the six metal coupons listed above. In contrast, bacterial survival was reduced only two to three orders of magnitude for spore layers on astroquartz and graphite composite exposed to 1 h of Mars UV irradiation. Scanning electron microscopy images of the bacterial monolayers on all eight spacecraft materials revealed that endospores of B. subtilis formed large aggregates of multilayered spores on astroquartz and graphite composite, but not on the other six spacecraft materials. It is likely that the formation of multilayered aggregates of endospores on astroquartz and graphite composite is responsible for the enhanced survival of bacterial cells on these materials.

Most autotransporter passenger domains, regardless of their diversity in function, fold or are predicted to fold as right-handed β-helices carrying various loops that are presumed to confer functionality. Our goal here was to identify the subdomain (loop) or amino acid sequence of the Pet passenger domain involved in the receptor binding site on the host cell for Pet endocytosis. Here, we show that d1 and d2 subdomains, as well as the amino acid sequence linking the subdomain d2 and the adjacent β-helix (PDWET), are not required for Pet secretion through the autotransporter system and that none of our deletion mutants altered the predicted long right-handed β-helical structure. Interestingly, Pet lacking the d2 domain (PetΔd2) was unable to bind on the epithelial cell surface, in contrast to Pet lacking d1 (PetΔd1) subdomain or PDWET sequences. Moreover, the purified d1 subdomain, the biggest subdomain (29.8 kDa) containing the serine protease domain, was also unable to bind the cell surface. Thus, d2 sequence (54 residues without the PDWET sequence) was required for Pet binding to eukaryotic cells. In addition, this d2 sequence was also needed for Pet internalization but not for inducing cell damage. In contrast, PetΔd1, which was able to bind and internalize inside the cell, was unable to cause cell damage. Furthermore, unlike Pet, PetΔd2 was unable to bind cytokeratin 8, a Pet receptor. These data indicate that the surface d2 subdomain is essential for the ligand-receptor (Pet-Ck8) interaction for Pet uptake and to start the epithelial cell damage by this toxin. PMID:27113356

Soluble proteins are often highly unstable under mixing conditions that involve dynamic contacting between the main liquid phase and a gas phase. The recombinant human growth hormone (rhGH) was recently shown to undergo aggregation into micrometer-sized solid particles composed of non-native (mis- or unfolded) protein, once its solutions were stirred or shaken to generate a continuously renewed air-water interface. To gain deepened understanding and improved quantification of the air-water interface effect on rhGH stability, we analyzed the protein's aggregation rate (r(agg)) at controlled specific air-water surface areas (a(G/L)) established by stirring or bubble aeration. We show that in spite of comparable time-averaged values for a(G/L) (≈ 100 m(2)/m(3)), aeration gave a 40-fold higher r(agg) than stirring. The enhanced r(agg) under aeration was ascribed to faster macroscopic regeneration of free a(G/L) during aeration as compared to stirring. We also show that r(agg) was independent of the rhGH concentration in the range 0.67 - 6.7 mg/mL, and that it increased linearly dependent on the available a(G/L). The nonionic surfactant Pluronic F-68, added in 1.6-fold molar excess over rhGH present, resulted in complete suppression of r(agg). Foam formation was not a factor influencing r(agg). Using analysis by circular dichroism spectroscopy and small-angle X-ray scattering, we show that in the presence of Pluronic F-68 under both stirring and aeration, the soluble protein retained its original fold, featuring native-like relative composition of secondary structural elements. We further provide evidence that the efficacy of Pluronic F-68 resulted from direct, probably hydrophobic protein-surfactant interactions that prevented rhGH from becoming attached to the air-water interface. Surface-induced aggregation of rhGH is suggested to involve desorption of non-native protein from the air-water interface as the key limiting step. Proteins or protein aggregates released

In order to minimize the forward contamination of Mars, spacecraft are assembled under cleanroom conditions that require several procedures to clean and sterilize components. Surface characteristics of spacecraft materials may contribute to microbial survival on the surface of Mars by protecting spores from sterilizing agents, including UV irradiation. The primary objective of this study was to evaluate the effects of surface characteristics of several spacecraft materials on the survival of Bacillus subtilis spores under simulated Martian conditions.

The authors used an immunogold labeling procedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage of platelet adhesion, a typical segregation of receptors occurred. Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IIIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIIb-IIIa, GPIV and p24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets, whereas GPIb was absent from pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized. During the early stages of platelet activation, surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranular Fg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric alpha-granular localization. At later stages of activation and especially after thrombin stimulation, no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were still present inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased over the granulomere and extended to the cell periphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed

In agricultural areas the soil erosion and soil loss estimation is vital information in long-term planning. During the initial period of the erosion a part of the soil particles and aggregates get transportable and nutrients and organic matter could be transported due to the effect of water or wind. This preliminary phase was studied with laboratory-scale rainfall simulator. Developed surface crust and aggregate size composition of the runoff was examined in six different slope-roughness-moisture content combination of a Cambisol and a Regosol. The ratio of micro- and macro aggregates in the runoff indicate the stability of the aggregates and determine the transport capacity of the runoff. Both soil samples were taken from field where the water erosion is a potential hazard. During the experiment the whole amount of runoff and sediment was collected through sieve series to a bucket to separate the micro- and macro aggregates. In case of both samples the micro aggregates dominate in the runoff and the runoff rates are similar. Although the runoff of the Regosol - with dominant >1000μm macro aggregate content - contained almost nothing but <50μm sized micro aggregates. Meanwhile the runoff of the Cambisol - with more balanced micro and macro aggregate content - contained dominantly 50-250μm sized micro aggregates and in some case remarkable ratio 250-1000μm sized macro aggregates. This difference occurred because the samples are resistant against drop erosion differently. In case of both sample the selectivity of the erosion and substance matrix redistribution manifested in mineral crusts in the surface where the quartz deposited in place while the lighter organic matter transported with the sediment. The detachment of the aggregates and the redistribution of the particles highly effect on the aggregate composition of the runoff which is connected with the quality of the soil loss. So while the estimation of soil loss quantity is more or less is easy, measuring

Aging-related emotional memory deficit is a well-known complication in Alzheimer's disease and normal aging. However, little is known about its molecular mechanism. To address this issue, we examined the role of norepinephrine (NE) and its relevant drug desipramine in the regulation of hippocampal long-term potentiation (LTP), surface expression of AMPA receptor, and associative fear memory in rats. We found that there was a defective regulation of NE content and AMPA receptor trafficking during fear conditioning, which were accompanied by impaired emotional memory and LTP in aged rats. Furthermore, we also found that the exogenous upregulation of NE ameliorated the impairment of LTP and emotional memory via enhancing AMPA receptor trafficking in aged rats, and the downregulation of NE impaired LTP in adult rats. Finally, acute treatment with NE or desipramine rescued the impaired emotional memory in aged rats. These results imply a pivotal role for NE in synaptic plasticity and associative fear memory in aging rats and suggest that desipramine is a potential candidate for treating aging-related emotional memory deficit.

While the aggregation and mass settlement of diatoms at the termination of blooms results in significant export of carbon from the surface ocean, the mechanisms of bloom aggregation have been poorly understood. The aggregation of a multispecies diatom bloom was investigated under controlled conditions in a 1200 liter, nutrient-enriched, laboratory mesocosm in order to elucidate the parameters sufficient to accurately predict bloom aggregation. A diverse bloom of diatoms dominated by several species of Chaetoceros and Thalassiosira progressed through a classic pattern of exponential, stationary, and senescent phases in the mesocosm. Aggregates larger than 0.5 mm became detectable on the eighth day after inoculation, and aggregates >1 mm increased exponentially from Day 10 onward producing the appearance of a mass aggregation event late on Day 10. The bloom aggregated sequentially with Thalassiosira dominating early aggregates and Chaetoceros dominating later ones. Chaetoceros resting spores formed only in aggregates. Aggregation was not linked to nutrient depletion or to the physiological state of the cells since the onset of aggregation and the mass aggregation event occurred 1 to 3 days prior to nutrient depletion and while carbon:nitrogen ratios of cells were still very low and growth rates high. Moreover, visible aggregates did not form in the mesocosm until cell abundances were considerably higher than abundances observed to aggregate in nature, suggesting that aggregation was not strongly linked to phytoplankton cell concentration. Complementary studies in this volume clarify the role of non-phytoplankton particles in aggregation of the mesocosm bloom. The mesocosm approach proved highly effective in producing an aggregating diatom bloom under controlled conditions.

Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate that the 949 phages target cell wall phospho-polysaccharides as their receptors, accounting for the specificity of the interactions of these phages with their lactococcal hosts. Such information may ultimately aid in the identification of strains/strain blends that do not present the necessary saccharidic target for infection by these problematic phages. PMID:25746988

Ferrihydrite is an abundant iron(oxy)hydroxide in soils and sediments and plays an important role in microbial iron cycling due to its high reactivity. Therefore, it is often synthesized and used in geomicrobiological and mineralogical studies. The reactivities of synthetic ferrihydrites vary between different studies and synthesis protocols. Hence, we synthesized five different ferrihydrites and characterized them with XRD, FTIR, XPS, and BET specific surface area. The reactivity of the ferrihydrite samples towards ascorbic acid was examined and compared with microbial reduction rates by Geobacter sulfurreducens. FTIR and XRD results show the presence of secondary, higher crystalline iron oxide phases like goethite and akaganeite for two samples. Consequently, those samples revealed lower biotic and abiotic reduction rates compared to pure ferrihydrite. Comparison of reduction rates with the specific surface area of all ferrihydrites showed neither correlation with abiotic reductive dissolution nor with microbial reduction. Especially one sample, characterized by a very low aggregation state and presence of secondary minerals, revealed a poor reactivity. We speculate that apart from the occurring secondary minerals also the low aggregation state played an important role. Decreasing aggregation diminishes the amount of kinks and edges on the surfaces, which are produced at contact sites in aggregates. According to dissolution theories, dissolution mainly starts at those surface defects and slows down with decreasing amount of defects. Furthermore, the non-aggregated ferrihydrite is free of micropores, a further stimulant for dissolution. Independent repetitions of experiments and syntheses according to the same protocol but without formation of secondary minerals, confirmed the low reactivity of the non-aggregated ferrihydrite. In summary, our results indicate that a decreasing aggregation state of ferrihydrite to a certain size does increase the reactivity

Neurotrophin-mediated cell survival and differentiation of vertebrate neurons is caused by ligand-specific binding to the Trk family of tyrosine kinase receptors. However, sites in the neurotrophins responsible for the binding to Trk receptors and the mechanisms whereby this interaction results in receptor activation and biological activity are unknown. Here we show that in nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), discontinuous stretches of amino acid residues group together on one side of the neurotrophin dimer forming a continuous surface responsible for binding to and activation of TrkA and TrkB receptors. Two symmetrical surfaces are formed along the two-fold axis of the neurotrophin dimer providing a model for ligand-mediated receptor dimerization. Mutated neurotrophins inducing similar levels of receptor phosphorylation showed different biological activities, suggesting that structural differences in a ligand may result in dissimilar responses in a given tyrosine kinase receptor. Our results allowed us to combine structural elements from NGF, BDNF and neurotrophin-3 to engineer a pan-neurotrophin that efficiently activates all Trk receptors and displays multiple neurotrophic specificities. Images PMID:8508763

Described is a school project to investigate aggregations in flatworms which may be influenced by light intensity, temperature, and some form of chemical stimulus released by already aggregating flatworms. Such investigations could be adopted to suit many educational levels of science laboratory activities. (DS)

CD44 is a cell surface adhesion receptor that is highly expressed in many cancers and regulates metastasis via recruitment of CD44 to the cell surface. Its interaction with appropriate extracellular matrix ligands promotes the migration and invasion processes involved in metastases. It was originally identified as a receptor for hyaluronan or hyaluronic acid and later to several other ligands including, osteopontin (OPN), collagens, and matrix metalloproteinases. CD44 has also been identified as a marker for stem cells of several types. Beside standard CD44 (sCD44), variant (vCD44) isoforms of CD44 have been shown to be created by alternate splicing of the mRNA in several cancer. Addition of new exons into the extracellular domain near the transmembrane of sCD44 increases the tendency for expressing larger size vCD44 isoforms. Expression of certain vCD44 isoforms was linked with progression and metastasis of cancer cells as well as patient prognosis. The expression of CD44 isoforms can be correlated with tumor subtypes and be a marker of cancer stem cells. CD44 cleavage, shedding, and elevated levels of soluble CD44 in the serum of patients is a marker of tumor burden and metastasis in several cancers including colon and gastric cancer. Recent observations have shown that CD44 intracellular domain (CD44-ICD) is related to the metastatic potential of breast cancer cells. However, the underlying mechanisms need further elucidation. PMID:28326306

Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin-8 (IL-8) and leukotriene B4 (LTB4) constitute key mediators by binding to the surfacereceptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll-like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down-regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL-8-induced and LTB4-induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down-regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL-8 and LTB4 pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation. PMID:25100544

A host of diverse stress techniques was applied to a monoclonal antibody (IgG2) to yield protein particles with varying attributes and morphologies. Aggregated solutions were evaluated for percent aggregation, particle counts, size distribution, morphology, changes in secondary and tertiary structure, surface hydrophobicity, metal content, and reversibility. Chemical modifications were also identified in a separate report (Luo, Q., Joubert, M. K., Stevenson, R., Narhi, L. O., and Wypych, J. (2011) J. Biol. Chem. 286, 25134–25144). Aggregates were categorized into seven discrete classes, based on the traits described. Several additional molecules (from the IgG1 and IgG2 subtypes as well as intravenous IgG) were stressed and found to be defined with the same classification system. The mechanism of protein aggregation and the type of aggregate formed depends on the nature of the stress applied. Different IgG molecules appear to aggregate by a similar mechanism under the same applied stress. Aggregates created by harsh mechanical stress showed the largest number of subvisible particles, and the class generated by thermal stress displayed the largest number of visible particles. Most classes showed a disruption of the higher order structure, with the degree of disorder depending on the stress process. Particles in all classes (except thermal stress) were at least partially reversible upon dilution in pH 5 buffer. High copper content was detected in isolated metal-catalyzed aggregates, a stress previously shown to produce immunogenic aggregates. In conclusion, protein aggregates can be a very heterogeneous population, whose qualities are the result of the type of stress that was experienced. PMID:21454532

The complex pathway seen in patients with the systemic inflammatory response syndrome (SIRS) does not readily respond to mediator blockade. All such trials conducted in SIRS patients have shown no benefit in reducing mortality. We have shown experimentally that in sepsis, the administration of beta 2-adrenoceptor agonists reduces hepatic cellular injury, whereas administration of an alpha 1-adrenoceptor agonist increases hepatic cellular injury. Inflammatory mediators can cause a dose-related reversible change in target endothelial cells (ECs). There is a substantial body of literature describing the anti-inflammatory effects of beta 2-adrenoceptor agonists. They reduce both the increased permeability and the production of inflammatory mediators from ECs. Cellular transduction processes are involved when adrenergic receptor agonists modify either the anti-inflammatory or proinflammatory response to sepsis in ECs. Inflammatory mediators and alpha 1-adrenoceptor agonists stimulate their trimeric G protein-linked receptors to produce diacylglycerol (DAG) and increase the intracellular concentration of calcium. DAG is involved in the production of both inflammatory proteins and lipids. In addition, mitogen-activated protein kinase (MAPK) is activated which is also involved in the production of inflammatory proteins and lipids. beta 2-adrenoceptor agonists activate their trimeric G protein-linked receptors to produce the stimulatory G protein (Gs). Gs stimulates adenyl cyclase to form cyclic adenosine monophosphate (cAMP) and activate protein kinase A (PKA). PKA is involved in activating gene transcription agents to produce anti-inflammatory proteins such as interleukin-10. PKA also inhibits phospholipase C and MAPK. Although promising, the use of beta-adrenoceptor agonists or agonists that increase cellular cAMP to activate the cells' endogenous anti-inflammatory pathway requires further study.

Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B−/− mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites. PMID:22740692

Affinity chromatography was used to identify a putative cell surfacereceptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.

Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surfacereceptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.

The initial step in retroviral infection involves specific interactions between viral envelope proteins (Env) and specific receptors on the surface of target cells. For many years, little was known about the entry receptors for HTLV-1. During this time, however, functional domains of the HTLV-1 Env were identified by analyzing the effects of neutralizing antibodies and specific mutations in Env on HTLV-1 infectivity. More recent studies have revealed that HTLV-1 infectivity involves interactions with three different molecules: heparan sulfate proteoglycans (HSPG), the VEGF-165 receptor Neuropilin 1 (NRP-1) and glucose transporter type 1 (GLUT1). Here, we revisit previously published data on the functional domains of Env in regard to the recent knowledge acquired about this multi-receptor complex. We also discuss the similarities and differences between HTLV-1 and other deltaretroviruses in regards to receptor usage.

A proper understanding of the behavior of dust particle aggregates immersed in a complex plasma first requires a knowledge of the basic properties of the system. Among the most important of these are the net electrostatic charge and higher multipole moments on the dust aggregate as well as the manner in which the aggregate interacts with the local electrostatic fields. The formation of elongated, fractal-like aggregates levitating in the sheath electric field of a weakly ionized RF generated plasma discharge has recently been observed experimentally. The resulting data has shown that as aggregates approach one another, they can both accelerate and rotate. At equilibrium, aggregates are observed to levitate with regular spacing, rotating about their long axis aligned parallel to the sheath electric field. Since gas drag tends to slow any such rotation, energy must be constantly fed into the system in order to sustain it. A numerical model designed to analyze this motion provides both the electrostatic charge and higher multipole moments of the aggregate while including the forces due to thermophoresis, neutral gas drag, and the ion wakefield. This model will be used to investigate the ambient conditions leading to the observed interactions. This research is funded by NSF Grant 1414523.

Three different ruthenium complexes have been synthesized and their luminescence properties in different solvent environments are reported. Luminescence intensities and excited state lifetimes of Ru-I, Ru-II and Ru-III vary with solvent viscosity. The excited state lifetime of Ru-I linearly increases in the viscosity range 1.76-12,100cP. Ru-II shows two linear increases: one in the low and another in the high viscosity ranges, whereas Ru-III illustrates a linear enhancement only in the low viscosity range. Interestingly, luminescence intensities and excited state lifetimes of Ru-I, Ru-II and Ru-III are found to be sensitive to nano-aggregation. However, the surfactant head charge and that of the ruthenium center as well as the hydrophobic tail of the ancillary ligand of the complexes have a great role in deciding the nature of the interaction and on the excited state properties at micellar surfaces. It is proposed that the long lifetime of Ru-III in water could be due to the coiling of the carbon chain of the ancillary ligand around the ruthenium center. At micelle surface, this coiling of the carbon chain is lost due to the parallel alignment with surfactants and thus quenching of the excited state lifetime is seen. Furthermore, it is shown that the variation of the excited state lifetime with respect to the change in surfactant concentration is a result of the formation of micelles from the surfactant monomer, thus, a novel technique for the determination of the critical micelle concentration (cmc) based on the long excited state lifetime of Ru-III located at the micellar nano-aggregates is reported.

Brain-derived neurotrophic factor (BDNF) plays a critical role in plasticity at glutamate synapses and the effects of repeated cocaine exposure. We recently showed that intracranial injection of BDNF into the rat nucleus accumbens (NAc), a key region for cocaine addiction, rapidly increases AMPA receptor (AMPAR) surface expression. To further characterize BDNF’s role in both rapid AMPAR trafficking and slower, homeostatic changes in AMPAR surface expression, we investigated the effects of acute (30 min) and long-term (24 h) treatment with BDNF on AMPAR distribution in NAc medium spiny neurons from postnatal rats co-cultured with mouse prefrontal cortex (PFC) neurons to restore excitatory inputs. Immunocytochemical studies showed that acute BDNF treatment increased cell surface GluA1 and GluA2 levels, as well as their co-localization, on NAc neurons. This effect of BDNF, confirmed using a protein crosslinking assay, was dependent on ERK but not AKT signaling. In contrast, long-term BDNF treatment decreased AMPAR surface expression on NAc neurons. Based on this latter result, we tested the hypothesis that BDNF plays a role in AMPAR “scaling down” in response to a prolonged increase in neuronal activity produced by bicuculline (24 h). Supporting this hypothesis, decreasing BDNF signaling with the extracellular BDNF scavenger TrkB-Fc prevented the scaling down of GluA1 and GluA2 surface levels in NAc neurons normally produced by bicuculline. In conclusion, BDNF exerts bidirectional effects on NAc AMPAR surface expression, depending on duration of exposure. Furthermore, BDNF’s involvement in synaptic scaling in the NAc differs from its previously described role in the visual cortex. PMID:24712995

This book is designed to help you understand our aggregate resources-their importance, where they come from, how they are processed for our use, the environmental concerns related to their mining and processing, how those concerns are addressed, and the policies and regulations designed to safeguard workers, neighbors, and the environment from the negative impacts of aggregate mining. We hope this understanding will help prepare you to be involved in decisions that need to be made-individually and as a society-to be good stewards of our aggregate resources and our living planet.

The development of methods to accurately quantify cell-surfacereceptors in living tissues would have a seminal impact in oncology. For example, accurate measures of receptor density in vivo could enhance early detection or surgical resection of tumors via protein-based contrast, allowing removal of cancer with high phenotype specificity. Alternatively, accurate receptor expression estimation could be used as a biomarker to guide patient-specific clinical oncology targeting of the same molecular pathway. Unfortunately, conventional molecular contrast-based imaging approaches are not well adapted to accurately estimating the nanomolar-level cell-surfacereceptor concentrations in tumors, as most images are dominated by nonspecific sources of contrast such as high vascular permeability and lymphatic inhibition. This article reviews approaches for overcoming these limitations based upon tracer kinetic modeling and the use of emerging protocols to estimate binding potential and the related receptor concentration. Methods such as using single time point imaging or a reference-tissue approach tend to have low accuracy in tumors, whereas paired-agent methods or advanced kinetic analyses are more promising to eliminate the dominance of interstitial space in the signals. Nuclear medicine and optical molecular imaging are the primary modalities used, as they have the nanomolar level sensitivity needed to quantify cell-surfacereceptor concentrations present in tissue, although each likely has a different clinical niche.

The development of methods to accurately quantify cell-surfacereceptors in living tissues would have a seminal impact in oncology. For example, accurate measures of receptor density in vivo could enhance early detection or surgical resection of tumors via protein-based contrast, allowing removal of cancer with high phenotype specificity. Alternatively, accurate receptor expression estimation could be used as a biomarker to guide patient-specific clinical oncology targeting of the same molecular pathway. Unfortunately, conventional molecular contrast-based imaging approaches are not well adapted to accurately estimating the nanomolar-level cell-surfacereceptor concentrations in tumors, as most images are dominated by nonspecific sources of contrast such as high vascular permeability and lymphatic inhibition. This article reviews approaches for overcoming these limitations based upon tracer kinetic modeling and the use of emerging protocols to estimate binding potential and the related receptor concentration. Methods such as using single time point imaging or a reference-tissue approach tend to have low accuracy in tumors, whereas paired-agent methods or advanced kinetic analyses are more promising to eliminate the dominance of interstitial space in the signals. Nuclear medicine and optical molecular imaging are the primary modalities used, as they have the nanomolar level sensitivity needed to quantify cell-surfacereceptor concentrations present in tissue, although each likely has a different clinical niche. PMID:26134619

Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in the toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.

The synthesized MgO nanoparticles were used to coat the light-weight expanded clay aggregates (LECA) and as a metronidazole (MNZ) adsorbent. X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier-transformed infrared (FTIR) techniques were employed to study the surface morphology and characteristics of the adsorbents. MgO/LECA clearly revealed the advantages of the nanocomposite particles, showing high specific surface area (76.12 m(2)/g), significant adsorption sites and functional groups. Between pH 5 and 9, the MNZ sorption was not significantly affected. Kinetic studies revealed that the MNZ adsorption closely followed the Avrami model, with no dominant process controlling the sorption rate. The study of the effects of foreign ions revealed that the addition of carbonate raised the MNZ removal efficiency of LECA by 8% and the total removal of MNZ by MgO/LECA. Furthermore, nitrate and hardness only marginally influenced the MNZ removal efficiency and their effects can be ranked in the order of carbonate>nitrate>hardness. The isotherm adsorption of MNZ was best fitted with the Langmuir model enlighten the monolayer MNZ adsorption on the homogeneous LECA and MgO/LECA surfaces. The maximum adsorption capacity under optimum conditions was enhanced from 56.31 to 84.55 mg/g for LECA and MgO/LECA, respectively. These findings demonstrated that the MgO/LECA nanocomposite showed potential as an efficient adsorbent for MNZ removal.

Functional activity of macrophages was studied in mice up to 15 days after intraperitoneal injection of 2.5 and 5.0 mg of fly ash using in vitro parameters. Fly ash did not cause any variation in the type of cellular response. The total cell number decreased significantly by 4 days after fly ash treatment but recovered subsequently. The decrease was dose dependent. Fly ash also caused a 50% depression in the FC receptor mediated phagocytosis of IgG coated sheep erythrocytes (SRBC) by macrophages at 2 days of dust treatment. However, the recovery began earlier with 2.5 mg fly ash than with 5.0 mg fly ash. These changes were not associated with any marked changes in esterase activity of macrophages following phagocytosis of fly ash.

Patchy colloids are attractive as programmable building blocks for metamaterials. Inverse patchy colloids, in which a charged surface is decorated with patches of the opposite charge, are additionally noteworthy as models for heterogeneously charged biological materials such as proteins. We study the phases and aggregation behavior of a single charged patch in an oppositely charged colloid with a single-site model. This single-patch inverse patchy colloid model shows a large number of phases when varying patch size. For large patch sizes we find ferroelectric crystals, while small patch sizes produce cross-linked gels. Intermediate values produce monodisperse clusters and unusual worm structures that preserve finite ratios of area to volume. The polarization observed at large patch sizes is robust under extreme disorder in patch size and shape. We examine phase-temperature dependence and coexistence curves and find that large patch sizes produce polarized liquids, in contrast to mean-field predictions. Finally, we introduce small numbers of unpatched charged colloids. These can either suppress or encourage aggregation depending on their concentration and the size of the patches on the patched colloids. These effects can be exploited to control aggregation and to measure effective patch size.

The cell surfacereceptor for ecotropic host-range (infection limited to mice or rats) murine leukemia viruses (MuLVs) is the widely expressed system y+ transporter for cationic amino acids (CAT-1). Like other retroviruses, ecotropic MuLV infection eliminates virus-binding sites from cell surfaces and results in complete interference to superinfection. Surprisingly, infection causes only partial (ca 40 to 60%) loss of mouse CAT-1 transporter activity. The NIH/Swiss mouse CAT-1 (mCAT-1) contains 622 amino acids with 14 hydrophobic potential membrane-spanning sequences, and it is known that the third extracellular loop from the amino terminus is required for virus binding. Although loop 3 is hypervariable in different species and mouse strains, consistent with its proposed role in virus-host coevolution, loop 3 sequences of both susceptible and resistant species contain consensus sites for N-linked glycosylation. Both of the consensus sites in loop 3 of mCAT-1 are known to be glycosylated and to contain oligosaccharides with diverse sizes (J. W. Kim and J. M. Cunningham, J. Biol. Chem. 268:16316-16320, 1993). We confirmed by several lines of evidence that N-linked glycosylation occludes a potentially functional virus-binding site in the CAT-1 protein of hamsters, thus contributing to resistance of that species. To study the role of receptor glycosylation in animals susceptible to infection, we eliminated loop 3 glycosylation sites by mutagenesis of an mCAT-1 cDNA clone, and we expressed wild-type and mutant receptors in mink fibroblasts and Xenopus oocytes. These receptors had indistinguishable transport properties, as determined by kinetic and voltage-jump electrophysiological studies of arginine uptake in oocytes and by analyses Of L-[3H]arginine uptake in mink cells. Bindings of ecotropic envelope glycoprotein gp7O to the accessible receptor sites on surfaces of mink cells expressing wild-type or mutant mCAT-1 were not significantly different in kinetics or in

The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design. PMID:26260789

The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design.

Myxococcus xanthus is a soil-dwelling bacterium that exhibits several fascinating collective behaviors including streaming, swarming, and generation of fruiting bodies. A striking feature of M. xanthus is that it periodically reverses its motility direction. The first stage of fruiting body formation is characterized by the aggregation of cells on a surface into round mesoscopic structures. Experiments have shown that this aggregation relies heavily on regulation of the reversal rate and local mechanical interactions, suggesting motility-induced phase separation may play an important role. We have adapted self-propelled particle models to include cell reversal and motility suppression resulting from sporulation observed in aggregates. Using 2D molecular dynamics simulations, we map the phase behavior in the space of Péclet number and local density and examine the kinetics of aggregation for comparison to experiments.

Viking observations suggest abundant silt and clay particles on Mars. It is proposed that some of these particles agglomerate to form sand size aggregates that are redeposited as sandlike features such as drifts and dunes. Although the binding for the aggregates could include salt cementation or other mechanisms, electrostatic bonding is considered to be a primary force holding the aggregates together. Various laboratory experiments conducted since the 19th century, and as reported here for simulated Martian conditions, show that both the magnitude and sign of electrical charges on windblown particles are functions of particle velocity, shape and composition, atmospheric pressure, atmospheric composition and other factors. Electrical charges have been measured for saltating particles in the wind tunnel and in the field, on the surfaces of sand dunes, and within dust clouds on earth. Similar, and perhaps even greater, charges are proposed to occur on Mars, which could form aggregates of silt and clay size particles

The surface density of the triggering receptors (e.g. NKp46 and NKp30) responsible for natural killer (NK) cell-mediated cytotoxicity determines the ability of NK cells to kill susceptible target cells. In this study, we show that prolactin up-regulates and cortisol down-regulates the surface expression of NKp46 and NKp30. The prolactin-mediated activation and the cortisol-mediated inhibition of natural cytotoxicity receptor (NCR) surface expression reflects gene regulation at the transcriptional level. NKp46 and NKp30 are the major receptors involved in the NK-mediated killing of K562, a human chronic myelogenous leukaemia cell line. Accordingly, the prolactin dramatically increased the NK-mediated killing of the K562 cell line, whereas cortisol abolished this activity. Our data suggest a mechanism by which prolactin activates the lytic function of NK cells, and cortisol inhibits the NK-mediated attack.

The direction and scope of the Office of Naval Research's Marine Aggregate Dynamics Accelerated Research Initiative will be the topic of an open-house style meeting February 14, 7:30-10:00 P.M. in Ballroom D of the Hyatt Regency New Orleans at the Louisiana Superdome. This meeting is scheduled during the AGU/American Society of Limnology and Oceanography Ocean Sciences Meeting February 12-16 in New Orleans.The critical focus of the ARI is the measurement and modeling of the dynamics of the biological, physical, chemical and molecular processes that drive aggregation and produce aggregates. This new ARI will provide funding in Fiscal Years 1991-1995 to identify and quantify mechanisms that determine the distribution, abundance and size spectrum of aggregated particulate matter in the ocean.

To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

The arithmetic processes of aggregation and averaging are basic to quantitative investigations of employment, unemployment, and related concepts. In explaining these concepts, this report stresses need for accuracy and consistency in measurements, and describes tools for analyzing alternative measures. (BH)

Platelet function hyper-activity has been reported in Dirofilaria immitis (heartworm, HW)-infected dogs. Although the mechanism of increased platelet hyper-activity has not yet been elucidated, it is suggested to be mediated by unknown factors, which may be related to adult HW components. This study aims to determine whether adult male HW whole body extract induces canine platelet aggregation in vitro. The results indicate that HW extract caused an aggregation of canine platelets in a concentration-dependent manner. This aggregation ability of the HW extract was not mediated by the adenosine diphosphate receptor. In addition, the mechanisms of aggregation did not require cyclooxygenase-dependent pathways, and the aggregating activity of substances contained in the HW extract was heat stable; therefore, the active substances may be different from collagen. Furthermore, the platelet aggregating activity remained within the molecular weight (MW)≥100,000 fraction obtained by ultrafiltrating the HW extract. In contrast, the MW <100,000 fraction also had a platelet aggregation ability, but the aggregation pattern was reversible and the maximum extent decreased, compared with the MW≥100,000 fraction response. Our experiments have been conducted using a whole body extract from adult HWs to determine with certainty the aggregating activity of HW elements on canine platelets. More studies are necessary to evaluate the effects of the metabolic products released from live adult worms in pulmonary arteries and the symbiont bacterium Wolbachia-derived antigens on canine platelet aggregation. PMID:28049921

Platelet function hyper-activity has been reported in Dirofilaria immitis (heartworm, HW)-infected dogs. Although the mechanism of increased platelet hyper-activity has not yet been elucidated, it is suggested to be mediated by unknown factors, which may be related to adult HW components. This study aims to determine whether adult male HW whole body extract induces canine platelet aggregation in vitro. The results indicate that HW extract caused an aggregation of canine platelets in a concentration-dependent manner. This aggregation ability of the HW extract was not mediated by the adenosine diphosphate receptor. In addition, the mechanisms of aggregation did not require cyclooxygenase-dependent pathways, and the aggregating activity of substances contained in the HW extract was heat stable; therefore, the active substances may be different from collagen. Furthermore, the platelet aggregating activity remained within the molecular weight (MW)≥100,000 fraction obtained by ultrafiltrating the HW extract. In contrast, the MW <100,000 fraction also had a platelet aggregation ability, but the aggregation pattern was reversible and the maximum extent decreased, compared with the MW≥100,000 fraction response. Our experiments have been conducted using a whole body extract from adult HWs to determine with certainty the aggregating activity of HW elements on canine platelets. More studies are necessary to evaluate the effects of the metabolic products released from live adult worms in pulmonary arteries and the symbiont bacterium Wolbachia-derived antigens on canine platelet aggregation.

Met, the tyrosine kinase receptor for the hepatocyte growth factor is a prominent regulator of cancer cell invasiveness and has emerged as a promising therapeutic target. Binding of the anti-Met monoclonal antibody DN30 to its epitope induces the proteolytic cleavage of Met, thereby impairing the invasive growth of tumors. The molecular mechanism controlling this therapeutic shedding process has so far been unknown. Here, we report that A Disintegrin And Metalloproteinase (ADAM)-10, but not ADAM-17, is required for DN30-induced Met shedding. Knockdown of ADAM-10 in different tumor cell lines or abrogation of its proteolytic activity by natural or synthetic inhibitors abolished Met down-regulation on the cell surface as well as reduction of Met activation. Moreover, hepatocyte growth factor-induced tumor cell migration and invasion were impaired upon ADAM-10 knockdown. Thus, the therapeutic effect of DN30 involves ADAM-10-dependent Met shedding, linking for the first time a specific metalloprotease to target therapy against a receptor tyrosine kinase. PMID:20554517

Leptospirosis is zoonotic and emerging infectious disease of global importance. Little is understood about Leptospira pathogenesis and host immune response. In the present work we have investigated how Leptospira modulates the host innate immune response mediated by Toll-like receptors (TLRs) via surface exposed proteins. We screened Leptospira outer membrane/surface proteins for their ability to activate/inhibit TLR2/4 signaling in HEK293 cell lines. Of these the 21 kDa Leptospira surface adhesin, Lsa21 had strong TLR2 and TLR4 activity leading to production of proinflammatory cytokines and expression of costimulatory molecules in mouse macrophages. This activity of Lsa21 on innate response was dependent on activation of mitogen activated protein kinases (MAPKs) via stimulating the rapid phosphorylation of p38, JNK and activation of transcription factor NF-κB. Additionally, neutralizing antibodies against TLR2 and TLR4 significantly inhibited cytokine secretion and attenuated Lsa21 induced phosphorylation of p38 and JNK. Furthermore, Lsa21 induced cytokine levels were significantly lower in TLR2−/− and TLR4−/− than in wild type mouse macrophage cell lines. Confocal microscopy and molecular docking confirmed that Lsa21 interacted with both TLR2 and TLR4. These results indicate that Lsa21 is a potent TLR2 and TLR4 agonist that induces strong innate response and may play important role in Leptospira pathogenesis. PMID:27996041

We previously reported that lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind NR5A nuclear receptors to regulate their activity. Here, the crystal structures of PIP2 and PIP3 bound to NR5A1 (SF-1) define a new interaction surface that is organized by the solvent-exposed PIPn headgroups. We find that stabilization by the PIP3 ligand propagates a signal that increases coactivator recruitment to SF-1, consistent with our earlier work showing that PIP3 increases SF-1 activity. This newly created surface harbors a cluster of human mutations that lead to endocrine disorders, thus explaining how these puzzling mutations cripple SF-1 activity. Finally, we propose that this new surface acts as a PIP3-regulated interface between SF-1 and coregulatory proteins, analogous to the function of membrane-bound phosphoinositides.

Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein's hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85 and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600 mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation.

Seasonal variability of soil properties measured in surface horizons of three soil types (Haplic Luvisol, Greyic Phaeozem, Haplic Cambisol) was studied in years 2007 and 2008. Undisturbed and disturbed soil samples were taken every month to evaluate field water content, bulk density, porosity, ration of gravitational and capillary pores, pHKCl and pHH2O, organic matter content and its quality, aggregate stability using WSA index. In addition, micromorphological features of soil aggregates were studied in thin soil sections that were made from undisturbed large soil aggregates. Results showed that soil aggregate stability depended on stage of the root zone development, soil management and climatic conditions. Larger aggregate stabilities and also larger ranges of measure values were obtained in the year 2007 then those measured in 2008. This was probably caused by lower precipitations and consequently lower soil water contents observed in 2007 than those measured in 2008. The highest aggregate stability was measured at the end of April in the years 2007 and 2008 in Haplic Luvisol and Greyic Phaeozem, and at the end of June in the year 2007 and at the beginning of June in 2008 in Haplic Cambisol. In all cases aggregate stability increased during the root growth and then gradually decreased due to summer rainfall events. Aggregate stability reflected aggregate structure and soil pore system development, which was documented on micromorphological images and evaluated using the ration of gravitational and capillary pores measured on the undisturbed sol samples. Acknowledgement: Authors acknowledge the financial support of the Grant Agency of the Czech Republic grant No. 526/08/0434, and the Ministry of Education, Youth and Sports grant No. MSM 6046070901.

Concurrent binding of ecdysterone immobilized on the nanodispersed magnetite with intact rat cells in the presence of free ecdysterone was investigated. The results imply the existence of high affinity ecdysterone-specific binding sites on the surface of liver and spleen macrophages, thymus and spleen lymphocytes, erythrocytes and hepatocytes. Membrane effects may be involved in the signal transduction mechanisms activated by ecdysterone.

GPRC6A is a G protein-coupled receptor activated by l-amino acids, which, based on analyses of knock-out mice, has been suggested to have physiological functions in metabolism and testicular function. The human ortholog is, however, mostly retained intracellularly in contrast to the cell surface-expressed murine and goldfish orthologs. The latter orthologs are Gq-coupled and lead to intracellular accumulation of inositol phosphates and calcium release. In the present study we cloned the bonobo chimpanzee GPRC6A receptor, which is 99% identical to the human receptor, and show that it is cell surface-expressed and functional. By analyses of chimeric human/mouse and human/bonobo receptors, bonobo receptor mutants, and the single nucleotide polymorphism database at NCBI, we identify an insertion/deletion variation in the third intracellular loop responsible for the intracellular retention and lack of function of the human ortholog. Genetic analyses of the 1000 genome database and the Inter99 cohort of 6,000 Danes establish the distribution of genotypes among ethnic groups, showing that the cell surface-expressed and functional variant is much more prevalent in the African population than in European and Asian populations and that this variant is partly linked with a stop codon early in the receptor sequence (rs6907580, amino acid position 57). In conclusion, our data solve a more than decade-old question of why the cloned human GPRC6A receptor is not cell surface-expressed and functional and provide a genetic framework to study human phenotypic traits in large genome sequencing projects linked with physiological measurement and biomarkers.

The aim of this study was to produce epithelial attachment on a typical implant abutment surface of smooth titanium. A challenging complication that hinders the success of dental implants is peri-implantitis. A common cause of peri-implantitis may results from the lack of epithelial sealing at the peri-implant collar. Histologically, epithelial sealing is recognized as the attachment of the basement membrane (BM). BM-attachment is promoted by activated platelet aggregates at surgical wound sites. On the other hand, platelets did not aggregate on smooth titanium, the surface typical of the implant abutment. We then hypothesized that epithelial BM-attachment was produced when titanium surface was modified to allow platelet aggregation. Titanium surfaces were coated with a protease activated receptor 4-activating peptide (PAR4-AP). PAR4-AP coating yielded rapid aggregation of platelets on the titanium surface. Platelet aggregates released robust amount of epithelial chemoattractants (IGF-I, TGF-β) and growth factors (EGF, VEGF) on the titanium surface. Human gingival epithelial cells, when they were co-cultured on the platelet aggregates, successfully attached to the PAR4-AP coated titanium surface with spread laminin5 positive BM and consecutive staining of the epithelial tight junction component ZO1, indicating the formation of complete epithelial sheet. These in-vitro results indicate the establishment of epithelial BM-attachment to the titanium surface. PMID:27741287

Soil aggregation was studied in a 21-year experiment conducted on an Anthrosol. The soil management regimes consisted of cropland abandonment, bare fallow without vegetation and cropping system. The cropping system was combined with the following nutrient management treatments: control (CONTROL, no nutrient input); nitrogen, phosphorus and potassium (NPK); straw plus NPK (SNPK); and manure (M) plus NPK (MNPK). Compared with the CONTROL treatment, the abandonment treatment significantly increased the formation of large soil macroaggregates (>2 mm) and consequently improved the stability of aggregates in the surface soil layer due to enhancement of hyphal length and of soil organic matter content. However, in response to long-term bare fallow treatment aggregate stability was low, as were the levels of aggregating agents. Long term fertilization significantly redistributed macroaggregates; this could be mainly ascribed to soil organic matter contributing to the formation of 0.5–2 mm classes of aggregates and a decrease in the formation of the >2 mm class of aggregates, especially in the MNPK treatment. Overall, hyphae represented a major aggregating agent in both of the systems tested, while soil organic compounds played significantly different roles in stabilizing aggregates in Anthrosol when the cropping system and the soil management regimes were compared.

Soil aggregation was studied in a 21-year experiment conducted on an Anthrosol. The soil management regimes consisted of cropland abandonment, bare fallow without vegetation and cropping system. The cropping system was combined with the following nutrient management treatments: control (CONTROL, no nutrient input); nitrogen, phosphorus and potassium (NPK); straw plus NPK (SNPK); and manure (M) plus NPK (MNPK). Compared with the CONTROL treatment, the abandonment treatment significantly increased the formation of large soil macroaggregates (>2 mm) and consequently improved the stability of aggregates in the surface soil layer due to enhancement of hyphal length and of soil organic matter content. However, in response to long-term bare fallow treatment aggregate stability was low, as were the levels of aggregating agents. Long term fertilization significantly redistributed macroaggregates; this could be mainly ascribed to soil organic matter contributing to the formation of 0.5–2 mm classes of